Compositions and methods for preventing the proliferation and epithelial-mesenchymal transition of epithelial cells

ABSTRACT

Compositions and preparations of fetal support tissue that prevent or reduce the proliferation and epithelial-mesenchymal transition (EMT) of epithelial cells, wherein the epithelial cells may be human epithelial cells and the human epithelial cells may be conjunctival, retinal, corneal, limbal, or renal epithelial cells. Methods of preventing or reducing the proliferation, cell migration, and EMT of epithelial cells in an individual in need thereof, wherein the epithelial cells may be human epithelial cells and the human epithelial cells may be conjunctival, retinal, corneal, limbal, or renal epithelial cells. Methods of preventing or treating proliferative vitreoretinopathy in an individual in need thereof.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.16/421,191, filed May 23, 2019, which is a continuation of U.S.application Ser. No. 15/160,487, filed May 20, 2016, which claims thebenefit of and right of priority to U.S. Provisional Application No.62/164,281 filed May 20, 2015, which is incorporated herein by referencein its entirety.

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, are methods for preventing orreducing proliferation, cell migration, or epithelial-mesenchymaltransition (EMT) of epithelial cells in an individual in need thereof,comprising: administering to the individual a therapeutically effectiveamount of a composition, comprising: (a) a preparation of fetal supporttissue; and (b) a pharmaceutically acceptable diluent, excipient,vehicle, or carrier, thereby preventing or reducing the proliferation,cell migration, or EMT of epithelial cells, wherein the epithelial cellsare not retinal pigment epithelial cells. In some embodiments, the EMTis associated with a disease or disorder other than proliferativevitreoretinopathy (PVR). In some embodiments, the EMT is associated witha disease or disorder selected from cancer, proliferative diabeticretinopathy, fibrotic lesion, and Retro-corneal membrane. In someembodiments, the fetal support tissue is selected from the groupconsisting of: placenta, placental amniotic membrane, umbilical cord,umbilical cord amniotic membrane, chorion, amnion-chorion, amnioticstroma, amniotic jelly, amniotic fluid, and a combination thereof. Insome embodiments, the fetal support tissue is frozen or previouslyfrozen. In some embodiments, the epithelial cells are selected fromconjunctival epithelial cells, corneal epithelial cells, limbalepithelial cells, and renal epithelial cells. In some embodiments, theepithelial cells are human epithelial cells. In some embodiments, thehuman epithelial cells are retinal pigment epithelial cells (RPE). Insome embodiments, the human epithelial cells are conjunctival epithelialcells. In some embodiments, the human epithelial cells are cornealepithelial cells. In some embodiments, the human epithelial cells arelimbal epithelial cells. In some embodiments, the human epithelial cellsare renal epithelial cells. In some embodiments, the preparation offetal support tissue is an extract of fetal support tissue, ahomogenate, a powder, morselized fetal support tissue, pulverized fetalsupport tissue, ground fetal support tissue, purified HC-HA/PTX3, or acombination thereof. In some embodiments, the composition is a gel, asolution, or a suspension. In some embodiments, the composition is in aninjectable form. In some embodiments, the preparation of fetal supporttissue comprises substantially isolated HC-HA/PTX3. In some embodiments,the preparation of fetal support tissue consists of substantiallyisolated HC-HA/PTX3. In some embodiments, the preparation of fetalsupport tissue comprises reconstituted HC-HA/PTX3. In some embodiments,the preparation of fetal support tissue comprises high molecular weighthyaluronan (HA) that is cross-linked by a covalent bond to the heavychain of inter-α-trypsin inhibitor (IαI), the high molecular weight HAhaving a molecular weight greater than 1000 kDa. In some embodiments,the preparation of fetal support tissue comprises pentraxin 3 (PTX-3).In some embodiments, the preparation of fetal support tissue comprisestumor necrosis factor-stimulated gene 6 protein (TSG-6). In someembodiments, the preparation of fetal support tissue comprisesthrombospondin-1 (TSP-1). In some embodiments, the ratio of totalprotein to HA in the composition is between 500 parts protein:1 part HAand 500 parts HA:1 parts protein. In some embodiments, the compositionprevents the proliferation and EMT of epithelial cells by counteractingthe actions of growth factors and cytokines. In some embodiments, thegrowth factors and cytokines are selected from the group consisting of:EGF, FGF-2, PDGF-A, PDGF-AB, PDGF-B, PDGF-C, TGF-β1, TGF-β2, TGF-β3,CTGF, HGF, IGF-1, G-CSF, IL-6, MCP-1, TNF-α, VEGF, and IFN-γ. In someembodiments, the composition further comprises an aqueous adjuvant. Insome embodiments, the composition is for local administration. In someembodiments the composition if formulated for injection. In someembodiments, the composition is formulated for intraocular injection,subretinal injection, intravitreal injection, periocular injection,subconjunctival injection, retrobulbar injection, intracameralinjection, or sub-Tenon's injection.

Disclosed herein, in certain embodiments, are methods method fortreating or preventing Proliferative Vitreoretinopathy (PVR) in anindividual in need thereof, comprising administering to the individual atherapeutically effective amount of an injectable composition,consisting essentially of: (a) substantially isolated HC-HA/PTX3,reconstituted HC-HA/PTX3, or a combination thereof; and (b) apharmaceutically acceptable diluent, excipient, vehicle, or carrier,thereby treating or preventing PVR. In some embodiments, the compositionconsists of: (a) substantially isolated HC-HA/PTX3, reconstitutedHC-HA/PTX3, or a combination thereof; and (b) a pharmaceuticallyacceptable diluent, excipient, vehicle, or carrier. In some embodiments,the composition consists of reconstituted HC-HA/PTX3 and apharmaceutically acceptable diluent, excipient, vehicle, or carrier. Insome embodiments, the composition consists of substantially isolatedHC-HA/PTX3 and a pharmaceutically acceptable diluent, excipient,vehicle, or carrier. In some embodiments, the substantially isolatedHC-HA/PTX3 is isolated from fetal support tissue is selected from thegroup consisting of: placenta, placental amniotic membrane, umbilicalcord, umbilical cord amniotic membrane, chorion, amnion-chorion,amniotic stroma, amniotic jelly, amniotic fluid, and a combinationthereof. In some embodiments, the fetal support tissue is frozen orpreviously frozen. In some embodiments, the fetal support tissue ishuman, non-human primate, bovine, or porcine. In some embodiments, thefetal support tissue is human. In some embodiments, the substantiallyisolated HC-HA/PTX3 is isolated from fetal support tissue byultracentrifugation. In some embodiments, the therapeutically effectiveamount is effective for preventing or reducing the proliferation, cellmigration or EMT of epithelial cells. In some embodiments, theepithelial cells are retinal pigment epithelial cells (RPE). In someembodiments, the epithelial cells are human epithelial cells. In someembodiments, the human epithelial cells are retinal epithelial cells. Insome embodiments, the injectable composition is a gel, a solution, or asuspension. In some embodiments, the composition comprises highmolecular weight hyaluronan (HA) that is cross-linked by a covalent bondto the heavy chain of inter-α-trypsin inhibitor (IαI), the highmolecular weight HA having a molecular weight greater than 1000 kDa. Insome embodiments, the composition comprises pentraxin 3 (PTX-3). In someembodiments, the composition comprises tumor necrosis factor-stimulatedgene 6 protein (TSG-6). In some embodiments, the ratio of total proteinto HA in the injectable composition is between 500 parts protein:1 partHA and 500 parts HA:1 parts protein. In some embodiments, the injectablecomposition prevents the proliferation and EMT of epithelial cells byinhibiting or suppressing the activity of one or more growth factors orcytokines. In some embodiments, the growth factors and cytokines areselected from the group consisting of: EGF, FGF-2, PDGF-A, PDGF-AB,PDGF-B, PDGF-C, TGFβ1, TGF-β2, TGF-β3, CTGF, HGF, IGF-1, G-CSF, IL-6,MCP-1, TNF-α, VEGF, and IFN-γ. In some embodiments, the injectablecomposition further comprises an aqueous adjuvant. In some embodiments,the injectable composition is for local administration. In someembodiments, the injectable composition is formulated for intraocularinjection, subretinal injection, intravitreal injection, periocularinjection, subconjunctival injection, retrobulbar injection,intracameral injection or sub-Tenon's injection. In some embodiments,the composition is formulated for intravitreal injection.

Disclosed herein, in certain embodiments, are methods method fortreating or preventing Proliferative Vitreoretinopathy (PVR) in anindividual in need thereof, comprising administering to the individual atherapeutically effective amount of an injectable composition,consisting essentially of: (a) substantially isolated HC-HA/PTX3,reconstituted HC-HA/PTX3, or a combination thereof (b) an additionaltherapeutic agent; and (c) a pharmaceutically acceptable diluent,excipient, vehicle, or carrier, thereby treating or preventing PVR. Insome embodiments, the composition consists of: (a) substantiallyisolated HC-HA/PTX3, reconstituted HC-HA/PTX3, or a combination thereof,(b) an additional therapeutic agent; and (c) a pharmaceuticallyacceptable diluent, excipient, vehicle, or carrier. In some embodiments,the additional therapeutic agent is an additional agent for treatingPVR. In some embodiments, the additional therapeutic agent is selectedfrom the group consisting of: oral Accutane, intravitreal triamcinoloneacetonide, ranibizumab, bevacizumab, dasatinib, pegaptanib sodium,N-acetyl-cysteine (NAC), pioglitazone, glucosamine, genistin,geldanamycin, fausdil, resveratrol, hepatocyte growth factor (HGF),BMP-7, LY-364947, diosgenin, emodin, pentoxyfilline, dipyridamole, aperoxisome proliferative-activated receptor-gamma (PPARγ) agonist, afemale sex hormone, and an antioxidant. In some embodiments, the femalesex hormone comprises estradiol or progesterone. In some embodiments,the antioxidant comprises beta carotene, vitamin C, vitamin E, lutein,zeaxanthin, and omega-3 fatty acids. In some embodiments the additionaltherapeutic agent is an additional agent for treating inflammation. Insome embodiments, the composition consists of: (a) substantiallyisolated HC-HA/PTX3, reconstituted HC-HA/PTX3, or a combination thereof;(b) an additional therapeutic agent; and (c) a pharmaceuticallyacceptable diluent, excipient, vehicle, or carrier. In some embodiments,the composition consists of reconstituted HC-HA/PTX3, an additionaltherapeutic agent, and a pharmaceutically acceptable diluent, excipient,vehicle, or carrier. In some embodiments, the composition consists ofsubstantially isolated HC-HA/PTX3, an additional therapeutic agent, anda pharmaceutically acceptable diluent, excipient, vehicle, or carrier.In some embodiments, the substantially isolated HC-HA/PTX3 is isolatedfrom fetal support tissue is selected from the group consisting of:placenta, placental amniotic membrane, umbilical cord, umbilical cordamniotic membrane, chorion, amnion-chorion, amniotic stroma, amnioticjelly, amniotic fluid, and a combination thereof. In some embodiments,the fetal support tissue is frozen or previously frozen. In someembodiments, the fetal support tissue is human, non-human primate,bovine, or porcine. In some embodiments, the fetal support tissue ishuman. In some embodiments, the substantially isolated HC-HA/PTX3 isisolated from fetal support tissue by ultracentrifugation. In someembodiments, the composition further comprises an additional therapeuticagent. In some embodiments, the therapeutically effective amount iseffective for preventing or reducing the proliferation, cell migrationor EMT of epithelial cells. In some embodiments, the epithelial cellsare retinal pigment epithelial cells (RPE). In some embodiments, theepithelial cells are human epithelial cells. In some embodiments, thehuman epithelial cells are retinal epithelial cells. In someembodiments, the injectable composition is a gel, a solution, or asuspension. In some embodiments, the composition comprises highmolecular weight hyaluronan (HA) that is cross-linked by a covalent bondto the heavy chain of inter-α-trypsin inhibitor (IαI), the highmolecular weight HA having a molecular weight greater than 1000 kDa. Insome embodiments, the composition comprises pentraxin 3 (PTX-3). In someembodiments, the composition comprises tumor necrosis factor-stimulatedgene 6 protein (TSG-6). In some embodiments, the preparation of fetalsupport tissue comprises thrombospondin-1 (TSP-1). In some embodiments,the ratio of total protein to HA in the injectable composition isbetween 500 parts protein:1 part HA and 500 parts HA:1 parts protein. Insome embodiments, the injectable composition prevents the proliferationand EMT of epithelial cells by inhibiting or suppressing the activity ofone or more growth factors or cytokines. In some embodiments, the growthfactors and cytokines are selected from the group consisting of: EGF,FGF-2, PDGF-A, PDGF-AB, PDGF-B, PDGF-C, TGFβ1, TGF-β2, TGF-β3, CTGF,HGF, IGF-1, G-CSF, IL-6, MCP-1, TNF-α, VEGF, and IFN-γ. In someembodiments, the injectable composition further comprises an aqueousadjuvant. In some embodiments, the injectable composition is for localadministration. In some embodiments, the injectable composition isformulated for intraocular injection, subretinal injection, intravitrealinjection, periocular injection, subconjunctival injection, retrobulbarinjection, intracameral injection or sub-Tenon's injection. In someembodiments, the composition is formulated for intravitreal injection.

Disclosed herein, in certain embodiments, are methods for treating orpreventing Proliferative Vitreoretinopathy (PVR) in an individual inneed thereof, comprising administering to the individual atherapeutically effective amount of an injectable composition,comprising: a preparation of fetal support tissue comprising HC-HA/PTX3and at least one other component of fetal support tissue; and apharmaceutically acceptable diluent, excipient, vehicle, or carrier,thereby treating or preventing PVR. In some embodiments, the fetalsupport tissue is placenta, placental amniotic membrane, umbilical cord,umbilical cord amniotic membrane, chorion, amnion-chorion, amnioticstroma, amniotic jelly, amniotic fluid, or a combination thereof. Insome embodiments, the fetal support tissue is frozen or previouslyfrozen. In some embodiments, the fetal support tissue is human,non-human primate, bovine, or porcine. In some embodiments, the fetalsupport tissue is human. In some embodiments, the therapeuticallyeffective amount is an amount effective for preventing or reducing theproliferation, cell migration or EMT of epithelial cells. In someembodiments, the epithelial cells are retinal pigment epithelial (RPE)cells. In some embodiments, the preparation of fetal support tissue isan extract of fetal support tissue, micronized fetal support tissue, ahomogenate, a powder, morselized fetal support tissue, pulverized fetalsupport tissue, ground fetal support tissue, purified HC-HA/PTX3, or acombination thereof. In some embodiments, the composition is a gel, asolution, or a suspension. In some embodiments, the composition isformulated for intraocular injection, subretinal injection, intravitrealinjection, periocular injection, subconjunctival injection, retrobulbarinjection, intracameral injection or sub-Tenon's injection.

Disclosed herein, in certain embodiments, are compositions forpreventing or reducing proliferation, cell migration, and/orepithelial-mesenchymal transition (EMT) of epithelial cells, comprising:(a) a preparation of fetal support tissue; and (b) a pharmaceuticallyacceptable diluent, excipient, vehicle, or carrier, wherein theepithelial cells are not retinal pigment epithelial cells. In someembodiments, the EMT is associated with a disease or disorder other thanproliferative vitreoretinopathy. In some embodiments, the EMT isassociated with a disease or disorder selected from cancer,proliferative diabetic retinopathy, fibrotic lesion, and Retro-cornealmembrane. In some embodiments, the fetal support tissue is selected fromthe group consisting of: placenta, placental amniotic membrane,umbilical cord, umbilical cord amniotic membrane, chorion,amnion-chorion, amniotic stroma, amniotic jelly, amniotic fluid, and acombination thereof. In some embodiments, the fetal support tissue isfrozen or previously frozen. In some embodiments, the fetal supporttissue is human, non-human primate, bovine, or porcine. In someembodiments, the fetal support tissue is human. In some embodiments, thecomposition is in a therapeutically effective amount for preventing orreducing the proliferation, cell migration or EMT of epithelial cells.In some embodiments, the epithelial cells are selected from conjunctivalepithelial cells, corneal epithelial cells, limbal epithelial cells, andrenal epithelial cells. In some embodiments, the epithelial cells arehuman epithelial cells. In some embodiments, the human epithelial cellsare retinal pigment epithelial cells (RPE). In some embodiments, thehuman epithelial cells are conjunctival epithelial cells. In someembodiments, the human epithelial cells are corneal epithelial cells. Insome embodiments, the human epithelial cells are limbal epithelialcells. In some embodiments, the human epithelial cells are renalepithelial cells. In some embodiments, the preparation of fetal supporttissue is an extract of fetal support tissue, micronized fetal supporttissue, a homogenate, a powder, morselized fetal support tissue,pulverized fetal support tissue, ground fetal support tissue, orpurified HC-HA/PTX3. In some embodiments, the composition is a gel, asolution, or a suspension. In some embodiments, the preparation of fetalsupport tissue comprises HC-HA/PTX3. In some embodiments, thepreparation of fetal support tissue comprises substantially isolatedHC-HA/PTX3. In some embodiments, the preparation of fetal support tissueconsists of substantially isolated HC-HA/PTX3. In some embodiments, thepreparation of fetal support tissue comprises reconstituted HC-HA/PTX3.In some embodiments, the preparation of fetal support tissue compriseshigh molecular weight hyaluronan (HA) that is cross-linked by a covalentbond to the heavy chain of inter-α-trypsin inhibitor (IαI), the highmolecular weight HA having a molecular weight greater than 1000 kDa. Insome embodiments, the preparation of fetal support tissue comprisespentraxin 3 (PTX-3). In some embodiments, the preparation of fetalsupport tissue comprises tumor necrosis factor-stimulated gene 6 protein(TSG-6). In some embodiments, the preparation of fetal support tissuecomprises thrombospondin-1 (TSP-1). In some embodiments, the ratio oftotal protein to HA in the injectable composition is between 500 partsprotein:1 part HA and 500 parts HA:1 parts protein. In some embodiments,the injectable composition prevents the proliferation and EMT ofepithelial cells by inhibiting the actions of growth factors andcytokines. In some embodiments, the growth factors and cytokines areselected from the group consisting of: EGF, FGF-2, PDGF-A, PDGF-AB,PDGF-B, PDGF-C, TGF-β1, TGF-β2, TGF-β3, CTGF, HGF, IGF-1, G-CSF, IL-6,MCP-1, TNF-α, VEGF and IFN-γ. In some embodiments, the injectablecomposition further comprises an aqueous adjuvant. In some embodiments,the injectable composition is for local administration. In someembodiments, the composition is formulated for injection. In someembodiments, the injectable composition is formulated for intraocularinjection, subretinal injection, intravitreal injection, periocularinjection, subconjunctival injection, retrobulbar injection,intracameral injection, or sub-Tenon's injection.

Disclosed herein, in certain embodiments, are injectable compositionsfor treating or preventing Proliferative Vitreoretinopathy (PVR),comprising: (a) substantially isolated HC-HA/PTX3, reconstitutedHC-HA/PTX3, or a combination thereof; and (b) a pharmaceuticallyacceptable diluent, excipient, vehicle, or carrier. In some embodiments,the composition consists of: (a) substantially isolated HC-HA/PTX3,reconstituted HC-HA/PTX3, or a combination thereof; and (b) apharmaceutically acceptable diluent, excipient, vehicle, or carrier. Insome embodiments, the composition consists of reconstituted HC-HA/PTX3and a pharmaceutically acceptable diluent, excipient, vehicle, orcarrier. In some embodiments, the composition consists of substantiallyisolated HC-HA/PTX3 and a pharmaceutically acceptable diluent,excipient, vehicle, or carrier. In some embodiments, the substantiallyisolated HC-HA/PTX3 is isolated from fetal support tissue is selectedfrom the group consisting of: placenta, placental amniotic membrane,umbilical cord, umbilical cord amniotic membrane, chorion,amnion-chorion, amniotic stroma, amniotic jelly, amniotic fluid, and acombination thereof. In some embodiments, the fetal support tissue isfrozen or previously frozen. In some embodiments, the fetal supporttissue is human, non-human primate, bovine, or porcine. In someembodiments, the fetal support tissue is human. In some embodiments, thesubstantially isolated HC-HA/PTX3 is isolated from fetal support tissueby ultracentrifugation. In some embodiments, the injectable compositionis in a therapeutically effective amount for preventing or reducing theproliferation, cell migration or EMT of epithelial cells. In someembodiments, the epithelial cells are retinal pigment epithelial cells(RPE). In some embodiments, the epithelial cells are human epithelialcells. In some embodiments, the human epithelial cells are retinalepithelial cells. In some embodiments, the injectable composition is agel, a solution, or a suspension. In some embodiments, the preparationof fetal support tissue comprises high molecular weight hyaluronan (HA)that is cross-linked by a covalent bond to the heavy chain ofinter-α-trypsin inhibitor (IαI), the high molecular weight HA having amolecular weight greater than 1000 kDa. In some embodiments, thepreparation of fetal support tissue comprises pentraxin 3 (PTX-3). Insome embodiments, the preparation of fetal support tissue comprisestumor necrosis factor-stimulated gene 6 protein (TSG-6). In someembodiments, the ratio of total protein to HA in the injectablecomposition is between 500 parts protein:1 part HA and 500 parts HA:1parts protein. In some embodiments, the injectable composition preventsthe proliferation and EMT of epithelial cells by inhibiting orsuppressing the activity of growth factors and/or cytokines. In someembodiments, the growth factors and cytokines are selected from thegroup consisting of: EGF, FGF-2, PDGF-A, PDGF-AB, PDGF-B, PDGF-C,TGF-β1, TGF-β2, TGF-β3, CTGF, HGF, IGF-1, G-CSF, IL-6, MCP-1, TNF-α,VEGF and IFN-γ. In some embodiments, the injectable composition furthercomprises an aqueous adjuvant. In some embodiments, the injectablecomposition is for local administration. In some embodiments, theinjectable composition is formulated for intraocular injection,subretinal injection, intravitreal injection, periocular injection,subconjunctival injection, retrobulbar injection, intracameralinjection, or sub-Tenon's injection.

Disclosed herein, in certain embodiments, are injectable compositionsfor treating or preventing Proliferative Vitreoretinopathy (PVR),consisting essentially of: (a) substantially isolated HC-HA/PTX3,reconstituted HC-HA/PTX3, or a combination thereof; (b) an additionaltherapeutic agent; and (c) a pharmaceutically acceptable diluent,excipient, vehicle, or carrier. In some embodiments, the compositionconsists of: (a) substantially isolated HC-HA/PTX3, reconstitutedHC-HA/PTX3, or a combination thereof; (b) an additional therapeuticagent; and (c) a pharmaceutically acceptable diluent, excipient,vehicle, or carrier. In some embodiments, the composition consists ofreconstituted HC-HA/PTX3, an additional therapeutic agent, and apharmaceutically acceptable diluent, excipient, vehicle, or carrier. Insome embodiments, the composition consists of substantially isolatedHC-HA/PTX3, an additional therapeutic agent, and a pharmaceuticallyacceptable diluent, excipient, vehicle, or carrier. In some embodiments,the substantially isolated HC-HA/PTX3 is isolated from fetal supporttissue is selected from the group consisting of: placenta, placentalamniotic membrane, umbilical cord, umbilical cord amniotic membrane,chorion, amnion-chorion, amniotic stroma, amniotic jelly, amnioticfluid, and a combination thereof. In some embodiments, the fetal supporttissue is frozen or previously frozen. In some embodiments, the fetalsupport tissue is human, non-human primate, bovine, or porcine. In someembodiments, the fetal support tissue is human. In some embodiments, thesubstantially isolated HC-HA/PTX3 is isolated from fetal support tissueby ultracentrifugation. In some embodiments, the additional therapeuticagent is selected from the group consisting of: oral Accutane,intravitreal triamcinolone acetonide, ranibizumab, bevacizumab,dasatinib, pegaptanib sodium, N-acetyl-cysteine (NAC), pioglitazone,glucosamine, genistin, geldanamycin, fausdil, resveratrol, hepatocytegrowth factor (HGF), BMP-7, LY-364947, diosgenin, emodin,pentoxyfilline, dipyridamole, a peroxisome proliferative-activatedreceptor-gamma (PPARγ) agonist, a female sex hormone, and anantioxidant. In some embodiments, the female sex hormone comprisesestradiol or progesterone. In some embodiments, the antioxidantcomprises beta carotene, vitamin C, vitamin E, lutein, zeaxanthin, andomega-3 fatty acids. In some embodiments, the injectable composition isin a therapeutically effective amount for preventing or reducing theproliferation, cell migration or EMT of epithelial cells. In someembodiments, the epithelial cells are retinal pigment epithelial cells(RPE). In some embodiments, the epithelial cells are human epithelialcells. In some embodiments, the human epithelial cells are retinalepithelial cells. In some embodiments, the injectable composition is agel, a solution, or a suspension. In some embodiments, the preparationof fetal support tissue comprises high molecular weight hyaluronan (HA)that is cross-linked by a covalent bond to the heavy chain ofinter-α-trypsin inhibitor (IαI), the high molecular weight HA having amolecular weight greater than 1000 kDa. In some embodiments, thepreparation of fetal support tissue comprises pentraxin 3 (PTX-3). Insome embodiments, the preparation of fetal support tissue comprisestumor necrosis factor-stimulated gene 6 protein (TSG-6). In someembodiments, the ratio of total protein to HA in the injectablecomposition is between 500 parts protein:1 part HA and 500 parts HA:1parts protein. In some embodiments, the injectable composition preventsthe proliferation and EMT of epithelial cells by inhibiting orsuppressing the activity of growth factors and/or cytokines. In someembodiments, the growth factors and cytokines are selected from thegroup consisting of: EGF, FGF-2, PDGF-A, PDGF-AB, PDGF-B, PDGF-C,TGF-β1, TGF-β2, TGF-β3, CTGF, HGF, IGF-1, G-CSF, IL-6, MCP-1, TNF-α,VEGF and IFN-γ. In some embodiments, the injectable composition furthercomprises an aqueous adjuvant. In some embodiments, the injectablecomposition is for local administration. In some embodiments, theinjectable composition is formulated for intraocular injection,subretinal injection, intravitreal injection, periocular injection,subconjunctival injection, retrobulbar injection, intracameralinjection, or sub-Tenon's injection.

Disclosed herein, in certain embodiments, are injectable compositionsfor treating or preventing Proliferative Vitreoretinopathy (PVR)comprising: a preparation of fetal support tissue comprising HC-HA/PTX3and at least one other component of fetal support tissue; and apharmaceutically acceptable diluent, excipient, vehicle, or carrier;wherein the composition is suitable for injection. In some embodiments,the fetal support tissue is placenta, placental amniotic membrane,umbilical cord, umbilical cord amniotic membrane, chorion,amnion-chorion, amniotic stroma, amniotic jelly, amniotic fluid, or acombination thereof. In some embodiments, the fetal support tissue isfrozen or previously frozen. In some embodiments, the fetal supporttissue is human, non-human primate, bovine, or porcine. In someembodiments, the fetal support tissue is human. In some embodiments, thecomposition is in an amount effective for preventing or reducing theproliferation, cell migration or EMT of epithelial cells. In someembodiments, the epithelial cells are retinal pigment epithelial (RPE)cells. In some embodiments, the preparation of fetal support tissue isan extract of fetal support tissue, micronized fetal support tissue, ahomogenate, a powder, morselized fetal support tissue, pulverized fetalsupport tissue, ground fetal support tissue, purified HC-HA/PTX3, or acombination thereof. In some embodiments, the composition is a gel, asolution, or a suspension. In some embodiments, the composition isformulated for intraocular injection, subretinal injection, intravitrealinjection, periocular injection, subconjunctival injection, retrobulbarinjection, intracameral injection or sub-Tenon's injection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the signaling pathways in the regulating of EMT withor without proliferation by growth factors.

FIGS. 2A-2D illustrate HC-HA/PTX3 formation and characterization ofHC-HA/PTX3 purified from human AME. FIG. 2A provides a schematicillustration of HC-HA/PTX3 formation. FIG. 2B illustrates HC-HA/PTX3purified from human AME. FIG. 2C illustrates that the HC-HA/PTX3purified from AME comprises HC1. FIG. 2D illustrates that HC-HA/PTX3purified from AME comprises PTX3.

FIGS. 3A-3B illustrate canonical but not non-canonical Wnt signaling issuppressed by immobilized HC-HA/PTX3 in LEPCs/LNCs. FIG. 3A illustratesthat HC-HA/PTX3 downregulates canonical Wnt signaling in human limbalepithelial progenitor cells (LEPCs) and niche cells (LNCs). FIG. 3Billustrates immunostaining of β-catenin and C-JUN seeded either onMatrigel or on immobilized HC-HA/PTX3.

FIGS. 4A-4D illustrate expression of TGF-β and TGF-β receptors in humancorneal fibroblasts (HCF). FIG. 4A illustrates TGF-β1 expression inHuman Corneal Fibroblasts (HCFs) seeded on plastic, HA, or HC-HA/PTX3,both with and without addition of exogenous TGF-β1. FIG. 4B illustratesTGF-β2 expression in HCFs seeded on plastic, HA, or HC-HA/PTX3, bothwith and without addition of exogenous TGF-β1. FIG. 4C illustratesTGF-β3 expression in HCFs seeded on plastic, HA, or HC-HA/PTX3, bothwith and without addition of exogenous TGF-β1. FIG. 4D exemplifies aNorthern blot showing expression of TGF-βRI, TGF-βRII, and TGF-βIII inHCFs seeded on plastic, HA, or HC-HA/PTX3, both with and withoutaddition of exogenous TGF-β1. FIG. 4E exemplifies nuclear translocationof pSmad2/3 cause by addition of exogenous TGF-β1. FIG. 4F exemplifiespositive cytoplasmic expression of α-SMA caused by addition of exogenousTGF-1.

FIGS. 5A-5C illustrate HC-HA/PTX3 inhibits proliferation in ARPE-19cells when stimulated with EGF+FGF-2. FIG. 5A illustrates HC-HA/PTX3does not affect the viability of normal ARPE-19 cells. FIG. 5Billustrates proliferation of ARPE-19 cells using immunostaining. 5Cillustrates proliferation of ARE-19 cells.

FIGS. 6A-6B illustrate HC-HA/PTX3 inhibits nuclear translocation ofpSmad2/3 in APRE-19 cells. FIG. 6A illustrates nuclear localization ofphosphorylated Smad2/3 using immunostaining. FIG. 6B illustrates nuclearlocalization of phosphorylated Smad2/3.

FIGS. 7A-7D illustrate development of PVR in rabbits. FIG. 7Aexemplifies fundus photographs of a normal rabbit eye without PVR. FIG.7B exemplifies a rabbit with tractional PVR four weeks after gasvitrectomy and intravitreal injection of RPE cells. FIG. 7C exemplifiesa cross-section of the normal rabbit eye without PVR after enucleation.FIG. 7D exemplifies a cross-section of the eye of the rabbit withtractional PVR with retinal detachment after enucleation.

FIG. 8 illustrates the effect of the addition of collagen gel (Col), AMextract AME, or collagen gel mixed with AM extract (Col+AME) on thesuppression of TGF-β promoter activity. BSA was used as a control.

FIG. 9 illustrates the effect of treatment with AME, HA, or HA+AME,compared to a control assay with BSA alone, on the suppression of TGF-βactivity. The promoter activity is displayed as relative luciferaseunits (RLU).

FIG. 10 illustrates the molecular weight ranges of hyaluronan in AMextracts separated by agarose gel electrophoresis. Amniotic membraneextracted by buffer A, B, C were treated with or without hyaluronidaseand electrophoretically separated by a 0.5% agarose gel.

FIG. 11 illustrates the molecular weight ranges of hyaluronan in AMextracts separated by agarose gel electrophoresis. Amniotic membraneextracted by buffer PBS were treated with or without hyaluronidase (10units/ml in Tris-HCl, pH 7.5, 150 mM NaCl) for 2 hr at 37° C. and runthrough 0.5% agarose gels. HA: positive hyaluronic acid control; L: AMextract after low speed centrifugation; H: AM extract after high speedcentrifugation.

FIG. 12 illustrates a western blot demonstrating that theinter-α-trypsin inhibitor (IαI) is present in AM extracts. IαI waspresent in AM extract A and C although the signal of bikunin was veryweak (.about.39 kDa). Prior to transfer to the western blot, the extractwas separated on a 4-15% denatured acrylamide gel.

FIG. 13 illustrates an immunoblot demonstrating that the inter-α-trypsininhibitor (IαI) is present in the AM extracts even after low (LS) orhigh speed (HS) centrifugation.

FIG. 14 illustrates an immunoblot of TSG-6 (Tumor NecrosisFactor-Stimulated Gene 6), either with (+) or without (−) hyaluronidasetreatment. The samples included total AM extract without centrifugation(T), AM Extract after extraction in isotonic low salt buffer (buffer A);high salt buffer (B); or 4 M guanidine HCl (C); as detailed in Example2. TSG-6 was present in the total extract, buffer A extract, and bufferC extract. The addition of hyaluronidase did not appear to alter theTSG-6 level in the extracts.

FIG. 15 illustrates an immunoblot analysis of the deglycosylation ofTSG-6 in AM. AM extract A, B, and C were treated with (+) or without 20units/ml PNGase F at 37° C. for 3 hours. Glycosylation of TSG-6 in AMwas then analyzed by western blot. The cell lysate of human cornealfibroblast (HCF) was used as a positive control.

FIG. 16 illustrates an immunoblot analysis of potential TSG-6 complexesin AM by digestion with Chondroitin Sulfate ABC lyase. AM extract A, B,and C were treated without (−) or with (+) 1 unit/ml ABC lyase at 37° C.for 2 hours. The possible disruption of TSG-6 complexes was thenanalyzed by western blot using an anti-TSG-6 antibody RAH-1:1:1000.

FIG. 17 illustrates an immunoblot of potential TSG-6 complexes in AM bydigestion with Chondroitin Sulfate ABC lyase. This is the sameexperiment as shown in FIG. 16 except that a different TSG-6 antibodywas used. Here, the anti-TSG-6 antibody was obtained from R & D Systems(cat #MAB2104).

FIG. 18 illustrates an immunoblot demonstrating the presence ofPentraxin (PTX3) in AM, using a rat monoclonal anti-PTX3 antibodyobtained from Alexis Biochemicals. HCF: human corneal fibroblast, T, A,B, C: AM extract Total, A, B, C, respectively; HAse: Hyaluronidase.

FIG. 19 illustrates an immunoblot demonstrating the presence of TSP-1 inAM. The monomeric TSP-1 (180 kDa) and the putative trimeric TSP-1 (540kDa) are indicated. The positive control, TSP-1, was purified from humanplatelets (Calbiochem, Cat #605225) and loaded as 100 ng/lane.

FIG. 20 illustrates an immunoblot demonstrating the presence of Smad 7in AM. AM was extracted with PBS or urea (2M urea in 50 mM Tris-HCl, pH7.5). 20 μg of total protein was loaded for each extract. Smad 7 wasdetected with goat anti-human Smad 7 (AF2029, 1:1000, R & D Systems).Smad 7 migrated as a band of ˜51 kDa.

FIG. 21 illustrates the number of migrated cells counted from six randommicroscopic fields (n=4, * indicates p<0.05 when compared withPBS+EGF+FGF-2+TGF-β1).

FIG. 22 illustrates the percentage of gel contraction compared amonggroups (n=4, * indicates p<0.05 compared with PBS+TGF-β1).

DETAILED DESCRIPTION OF THE INVENTION

The present application describes compositions and methods forpreventing or reducing the proliferation, cell migration, and/orepithelial-mesenchymal transition (EMT) of epithelial cells, wherein theepithelial cells are human epithelial cells and the human epithelialcells are selected from: retinal pigment epithelial, conjunctival,retinal, corneal, limbal, or renal epithelial cells. Additionally, thepresent application describes compositions and methods for theprevention and treatment of proliferative vitreoretinopathy in anindividual in need thereof.

It is known that proliferation, cell migration and EMT occur whenepithelial cells such as, for example, retinal pigment epithelial, humanconjunctival, retinal, corneal, limbal, or renal epithelial cells areexposed to growth factors and cytokines such as, for example, EGF,FGF-2, PDGF-A, PDGF-AB, PDGF-B, PDGF-C, TGF-β1, TGF-β2, TGF-β3, CTGF,HGF, IGF-1, G-CSF, IL-6, MCP-1, TNF-α, VEGF or IFN-γ and ethylene glycoltetraacetic acid (EGTA) either in vitro or in vivo.

Further, it is known that transplantation of cryopreserved amnioticmembrane (AM) tissue onto the ocular surface providesanti-proliferative, anti-inflammatory, anti-scarring and anti-angiogenicactions in both corneal and limbal epithelial cells to promote woundhealing.

What is needed is a composition that prevents or reduces proliferation,cell migration and EMT of epithelial cells, can be administered withoutthe need of surgical transplantation and can additionally beadministered to non-surface epithelial cells such as, for example,retinal and renal epithelial cells.

A description of certain embodiments follows. It will be understood thatthe particular embodiments of the application are shown by way ofillustration and not as limitations of the application.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. All patents, patentapplications, published, applications and publications, GENBANKsequences, websites and other published materials referred to throughoutthe entire disclosure herein, unless noted otherwise, are incorporatedby reference in their entirety. In the event that there is a pluralityof definitions for terms herein, those in this section prevail. Wherereference is made to a URL or other such identifier or address, it isunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information is known andcan be readily accessed, such as by searching the internet and/orappropriate databases. Reference thereto evidences the availability andpublic dissemination of such information.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence,“about 5 μg” means “about 5 μg” and also “5 μg.” Generally, the term“about” includes an amount that would be expected to be withinexperimental error.

As used herein, the terms “subject”, “individual”, and “patient” areused interchangeably. None of the terms are to be interpreted asrequiring the supervision of a medical professional (e.g., a doctor,nurse, physician's assistant, orderly, hospice worker). As used herein,the subject is any animal, including mammals (e.g., a human or non-humananimal) and non-mammals. In one embodiment of the methods andcompositions provided herein, the mammal is a human.

As used herein, the terms “treat,” “treating” or “treatment,” and othergrammatical equivalents, include: alleviating, abating or amelioratingone or more symptoms of a disease or condition. In some embodiments,treating is alleviating, abating or ameliorating one or more symptoms ofepithelial-mesenchymal transition. In some embodiments, treating isalleviating, abating or ameliorating one or more symptoms ofproliferative vitreoretinopathy. In some embodiments, treating isalleviating, abating or ameliorating one or more symptoms ofinflammation. In some embodiments, treating is preventing or reducingthe appearance, severity or frequency of one or more additional symptomsof a disease or condition. In some embodiments, the methods includepreventing or reducing the appearance, severity or frequency of one ormore additional symptoms of epithelial-mesenchymal transition. In someembodiments, the methods include preventing or reducing the appearance,severity or frequency of one or more additional symptoms ofproliferative vitreoretinopathy. In some embodiments, the methodsinclude preventing or reducing the appearance, severity or frequency ofone or more additional symptoms of inflammation. In some embodiments,the methods include ameliorating or preventing the underlying metaboliccauses of one or more symptoms of a disease or condition, inhibiting thedisease or condition, such as, for example, arresting the development ofthe disease or condition, relieving the disease or condition, causingregression of the disease or condition, relieving a condition caused bythe disease or condition, or inhibiting the symptoms of the disease orcondition either prophylactically and/or therapeutically.

As used herein, “fetal support tissue” means tissue used to support thedevelopment of a fetus. Examples of fetal support tissue include, butare not limited to, (i) placental amniotic membrane (PAM), orsubstantially isolated PAM, (ii) umbilical cord amniotic membrane (UCAM)or substantially isolated UCAM, (iii) chorion or substantially isolatedchorion, (iv) amnion-chorion or substantially isolated amnion-chorion,(v) amniotic stroma or substantially isolated amniotic stroma, (vi)placenta or substantially isolated placenta, (vii) umbilical cord orsubstantially isolated umbilical cord, (viii) amniotic fluid, or (ix)any combinations thereof. Fetal support tissue is also usedinterchangeably with “gestational tissue.” In some embodiments thegestational tissue is “mammalian gestational tissue” or “humangestational tissue (“HGT”).” In some embodiments, the fetal supporttissue is obtained from a mammal. In some embodiments, the fetal supporttissue is from human, non-human primate, cow, or pig. In someembodiments, the fetal support tissue is from human. In someembodiments, the fetal support tissue is ground, pulverized, morselized,a graft, a powder, a gel, a homogenate, or an extract. In someembodiments, the fetal support tissue is aseptically processed. In someembodiment, the fetal support tissue is terminally-sterilized.

As used herein, “placenta” means the organ that connects a developingfetus to the maternal uterine wall to allow nutrient uptake, wasteelimination, and gas exchange via the maternal blood supply. Theplacenta is composed of three layers. The innermost placental layersurrounding the fetus is called amnion. The allantois is the middlelayer of the placenta (derived from the embryonic hindgut); bloodvessels originating from the umbilicus traverse this membrane. Theoutermost layer of the placenta, the chorion, comes into contact withthe endometrium. The chorion and allantois fuse to form thechorioallantoic membrane.

As used herein, “chorion” means the membrane formed by extraembryonicmesoderm and the two layers of trophoblasts. The chorionic villi emergefrom the chorion, invade the endometrium, and allow transfer ofnutrients from the maternal blood to fetal blood. The chorion consistsof two layers: an outer layer formed by the trophoblast, and an innerlayer formed by the somatic mesoderm; the amnion is contact with thelatter. The trophoblast is made up of an internal layer of cubical orprismatic cells, the cytotrophoblast or layer of Langhans, and anexternal layer of richly nucleated protoplasm devoid of cell boundaries,the syncytiotrophobast. The avascular amnion is adherent to the innerlayer of the chorion.

As used herein, “amnion-chorion” means a product comprising amnion andchorion. In some embodiments, the amnion and the chorion are notseparated (i.e., the amnion is naturally adherent to the inner layer ofthe chorion). In some embodiments, the amnion is initially separatedfrom the chorion and later combined with the chorion during processing.

As used herein, “umbilical cord” means the organ that connects adeveloping fetus to the placenta. The umbilical cord is composed ofWharton's jelly, a gelatinous substance made largely formmucopolysaccharides. It contains one vein, which carries oxygenated,nutrient-rich blood to the fetus, and two arteries that carrydeoxygenated, nutrient-depleted blood away. In some embodiments, theblood vessels have been substantially removed from the umbilical cordtissue. In some embodiments, a portion of the Wharton's Jelly has beenremoved. In some embodiments, the blood vessels and a portion of theWharton's Jelly have been removed.

As used herein, “placental amniotic membrane” (PAM) means amnioticmembrane derived from the placenta. In some embodiments, the PAM issubstantially isolated.

As used herein, “umbilical cord amniotic membrane” (UCAM) means amnioticmembrane derived from the umbilical cord. UCAM is a translucentmembrane. The UCAM has multiple layers: an epithelial layer; a basementmembrane; a compact layer; a fibroblast layer; and a spongy layer. Itlacks blood vessels or a direct blood supply. In some embodiments, theUCAM is substantially isolated. In some embodiments, the UCAM comprisesall of the Wharton's Jelly. In some embodiments, the UCAM comprises aportion of the Wharton's Jelly. In some embodiments, the UCAM comprisesblood vessels and/or arteries. In some embodiments, the UCAM comprisesall of the Wharton's Jelly and blood vessels and/or arteries. In someembodiments, the UCAM comprises part of the Wharton's Jelly and bloodvessels and/or arteries.

As used herein, “substantially isolated” or “isolated” means that thefetal support tissue product has been separate from undesired materials(e.g., red blood cells, blood vessels, and arteries) derived from theoriginal source organism. Purity, or “isolation” may be assayed bystandard methods, and will ordinarily be at least about 10% pure, moreordinarily at least about 20% pure, generally at least about 30% pure,and more generally at least about 40% pure; in further embodiments atleast about 50% pure, or more often at least about 60% pure; in stillother embodiments, at least about 95% pure.

As used herein, “biological activity” means the activity of polypeptidesand polysaccharides. In some embodiments, the activity of polypeptidesand polysaccharides found in umbilical cord (and substantially isolatedumbilical cord), UCAM (and substantially isolated UCAM), placenta (andsubstantially isolated placenta), PAM (and substantially isolated PAM),chorion (and substantially isolated chorion), or amnion-chorion (andsubstantially isolated amnion-chorion). In some embodiments, thebiological activity is anti-scarring activity, anti-inflammationactivity, anti-angiogenic activity, and wound healing. In someembodiments, the biological activity is anti-inflammation activity. Insome embodiments, the biological activity comprises the biologicalactivity of a fetal support tissue preparation or composition. In someembodiments, the biological activity comprises the biological activityof HC-HA/PTX3.

As used herein, the substantial preservation of biological activity orstructural integrity means that when compared to the biological activityand structural integrity of non-processed tissue, the biologicalactivity and structural integrity of the fetal support tissue producthas only decreased by about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 50%, or about 60%.

As used herein, “freezing” refers to exposing the fetal support tissueproduct below about or at 0° C., −5° C., −10° C., −20° C., −40° C., −50°C., −60° C., −70° C., −80° C., −90° C., or −100° C. for a period of timeof about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, orlonger.

As used herein, “powder” means matter in the form of fine dry particlesor matrix. In some embodiments, the particles are not uniform in size.In some embodiments, the particles are substantially uniform in size.

As used herein, “grinding” means any method of reducing fetal supporttissue to small particles or a powder. The term grinding includesmicronizing, pulverizing, homogenizing, filing, milling, grating,pounding, and crushing.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disease or condition being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forexample, an “effective amount” for therapeutic uses is the amount of thecomposition including a compound as disclosed herein required to providea clinically significant decrease in disease symptoms without undueadverse side effects. An appropriate “effective amount” in anyindividual case may be determined using techniques, such as a doseescalation study. The term “therapeutically effective amount” includes,for example, a prophylactically effective amount. An “effective amount”of a compound disclosed herein, is an amount effective to achieve adesired effect or therapeutic improvement without undue adverse sideeffects. It is understood that “an effective amount” or “atherapeutically effective amount” can vary from individual toindividual, due to variation in metabolism of the injectablecomposition, age, weight, general condition of the individual, thecondition being treated, the severity of the condition being treated,and the judgment of the prescribing physician. In some embodiments, aneffective amount is an amount that prevents or reduces the symptoms ofPVR. In some embodiments, an effective amount is an amount that reduces,inhibits or prevents cell migration, cell proliferation and/or EMT ofepithelial cells.

Epithelial-mesenchymal transition (EMT) is a process by which epithelialcells lose their cell polarity and cell-cell adhesion, and gainmigratory and invasive properties. EMT occurs in processes such asmesoderm formation, neural tube formation, wound healing, as well as theinitiation of metastasis for cancer progression. EMT can be inducedthrough several signal signaling pathways, including TGF-β, FGF, EGF,HGF, Wnt/beta-catenin, and Notch.

Proliferative vitreoretinopathy (PVR) is a disease that develops as acomplication of rhegmatogenous retinal detachment. When fluid from thevitreous humor enters a hole in the retina and accumulates in thesubretinal space, the tractional force of the vitreous on the retina iswhat results in rhegmatogenous retinal detachment. During this processthe retinal cell layers come in contact with vitreous cytokines, whichcan trigger the retinal pigmented epithelium (RPE) to proliferate andmigrate. The RPE cells undergo epithelial-mesenchymal transition (EMT)and develop the ability to migrate out into the vitreous. Duringmigration of the RPE, these cells lay down fibrotic membranes whichcontract and pull at the retina, and can lead to secondary retinaldetachment after primary retinal detachment surgery.

Compositions

Disclosed herein, in certain embodiments, are compositions forpreventing or reducing proliferation, cell migration, and/orepithelial-mesenchymal transition (EMT) of epithelial cells, comprising:a preparation of fetal support tissue; and a pharmaceutically acceptablediluent, excipient, vehicle, or carrier. Further disclosed herein, incertain embodiments, are injectable compositions for preventing orreducing proliferative venous retinopathy (PVR) in an individual in needthereof, comprising: a preparation of fetal support tissue; and apharmaceutically acceptable diluent, excipient, vehicle, or carrier.Further disclosed herein, in certain embodiments, are injectablecompositions for preventing or reducing proliferative venous retinopathy(PVR) in an individual in need thereof, consisting essentially of:substantially isolated HC-HA/PTX3, reconstituted HC-HA/PTX3(rcHC-HA/PTX3); and a pharmaceutically acceptable diluent, excipient,vehicle, or carrier. Further disclosed herein, in certain embodiments,are injectable compositions for preventing or reducing proliferativevenous retinopathy (PVR) in an individual in need thereof, consistingessentially of: substantially isolated HC-HA/PTX3, reconstitutedHC-HA/PTX3 (rcHC-HA/PTX3); an additional therapeutic agent; and apharmaceutically acceptable diluent, excipient, vehicle, or carrier.

In some embodiments, the preparation of fetal support tissue comprisesHC-HA/PTX3. In some embodiments, the preparation of fetal support tissuecomprises: high molecular weight hyaluronan (HA) that is cross-linked bya covalent bond to the heavy chain of inter-α-trypsin inhibitor (IαI),the high molecular weight HA having a molecular weight greater than 1000kDa. In some embodiments, the preparation of fetal support tissuecomprises: pentraxin 3 (PTX-3, PTX3). In some embodiments, thepreparation of fetal support tissue comprises: tumor necrosisfactor-stimulated gene 6 protein (TSG-6). In some embodiments, thepreparation of fetal support tissue comprises: thrombospondin-1 (TSP-1).In some embodiments, the ratio of total protein to HA in the compositionis less than 500 parts protein:1 part HA. In some embodiments, the ratioof HA to total protein in the composition is less than 500 parts HA:1part protein. In some embodiments, the preparation of fetal supporttissue comprises HC-HA/PTX3 complex. In some embodiments, thepreparation of fetal support tissue comprises substantially purifiedHC-HA/PTX3 complex.

In some embodiments, the epithelial cells are human epithelial cells. Insome embodiments, the human epithelial cells are retinal pigmentepithelial cells (RPE). In some embodiments, the human epithelial cellsare corneal epithelial cells. In some embodiments, the human epithelialcells are limbal epithelial cells. In some embodiments, the humanepithelial cells are conjunctival epithelial cells. In some embodiments,the human epithelial cells are renal epithelial cells.

In some embodiments, the composition prevents the proliferation and EMTof epithelial cells by suppressing the activity of growth factors andcytokines. In some embodiments, the growth factors and cytokines areselected from the group consisting of: EGF, FGF-2, PDGF-A, PDGF-AB,PDGF-B, PDGF-C, TGF-β1, TGF-β2, TGF-β3, CTGF, HGF, IGF-1, G-CSF, IL-6,MCP-1, TNF-α, VEGF and IFN-γ. In some embodiments, the compositioninhibits signaling pathways in epithelial cells to inhibit proliferationand EMT. In some embodiments, the signaling pathways are canonical Wntsignaling and TGF-β-induced Smad/ZEB signaling.

In some embodiments, the composition comprises the preparation of fetalsupport tissue and a pharmaceutically acceptable diluent, excipient, orcarrier. In some embodiments, the composition further comprises anaqueous adjuvant. In some embodiments, the composition is for localadministration. In some embodiments, the composition is formulated forinjection. In some embodiments, the composition is formulated forintraocular injection, subretinal injection, intravitreal injection,periocular injection, subconjunctival injection, retrobulbar injection,intracameral injection or sub-Tenon's injection.

Preparations of Fetal Support Tissue

In some embodiments, the preparation of fetal support tissue comprisesplacental tissue, umbilical cord tissue, placental amniotic membranetissue, chorion tissue, amniotic stroma, amnion-chorion tissue, UCAMtissue, amniotic fluid, or combinations thereof. In some embodiments,the preparation of fetal support tissue is an extract of fetal supporttissue, micronized fetal support tissue, a homogenate of fetal supporttissue, a powder of fetal support tissue, morselized fetal supporttissue, pulverized fetal support tissue, ground fetal support tissue,purified HC-HA/PTX3, or a combination thereof. In some embodiments, thepreparation of fetal support tissue is prepared from fresh, frozen orpreviously frozen fetal support tissue. In some embodiments, thepreparation of fetal support tissue is prepared from frozen orpreviously frozen of fetal support tissue. In some embodiments, thepreparation of fetal support tissue comprises HA, IαI, TSG-6, PTX-3,TSP-1, or a combination thereof. In some embodiments, the preparation offetal support tissue comprises HC-HA/PTX3 complex. In some embodiments,the preparation of fetal support tissue comprises purified HC-HA/PTX3.In some embodiments, the preparation of fetal support tissue comprisesultracentrifuged HC-HA/PTX3. In some embodiments, the preparation offetal support tissue consists of purified HC-HA/PTX3. In someembodiments, the preparation of fetal support tissue comprisesreconstituted HC-HA/PTX3.

In some embodiments, the preparation of fetal support tissue suppressesTGF-β promoter activity; increases apoptosis in macrophages; decreasesproliferation, decreases migration, and increases apoptosis of humanvascular endothelial cells; decreases viability of human fibroblasts;decreases inflammation; and prevents apoptosis of epithelial cellsexposed to storage and injury. In some embodiments, the preparations offetal support tissue and injectable compositions described herein areused to treat diseases related to TGF-β upregulation, such asangiogenesis, wound healing, and tissue inflammation.

TGF-β is the prototypic cytokine that is involved in tissueinflammation, in addition to wound healing and scar formation. Mammaliancells express three different TGF-βs: TGF-β1, TGF-β2, and TGFβ3. TGF-βis the most potent cytokine promoting myofibroblast differentiation byup-regulating expression of α-SMA, integrin α5β1, and EDAdomain-containing fibronectin (Fn) in a number of cell types, includingfibroblasts. TGF-β also up-regulates the expression of such matrixcomponents as collagens and proteoglycans, down-regulates proteinase andmatrix metalloproteinases, and up-regulates their inhibitors.Collectively, these actions result in increased cell-matrix interactionsand adhesiveness, as well as deposition and formation of scar tissue.

TGF-βs exert their actions via binding with TGF-β receptors (TGF-βRs) onthe cell membrane. In human cells, there are three TGF-βRs, namelyTGF-βR type I (TGF-βRI), type II (TGF-βRII), and type III (TGF-βRIII).TGF-βs, serving as ligands, bind with a serine, threonine kinasereceptor complex made of TGF-βRI and TGF-βRII; such a binding isfacilitated by TGF-βRIII, which is not a serine, threonine kinasereceptor. Binding with TGF-βRII activates TGF-βRI, which is responsiblefor direct phosphorylation of a family of effector proteins known asSmads, which modulate transcription of a number of target genes,including those described herein, participating in scar formation.

Suppression of TGF-β can be achieved by neutralizing antibodies to TGF-βand agents that intercede the signaling mediated by TGF-β such asdecorin. Most of the literature has shown suppression of TGF-β beingachieved at the level of modulating the TGF-β activation, binding withits receptor, or its signal transduction. It has been shown thatamniotic membrane can achieve such an inhibition at the level oftranscription, i.e., to turn off transcription of TGF-β1 genes. Inparticular, amniotic membrane has been shown to suppress TGF-β signalingin human corneal and limbal fibroblasts, and human conjunctival andpterygium body fibroblasts.

Hyaluronic acid (HA) is a natural sugar found in the synovial jointfluid, the vitreous humor of the eye, the cartilage, blood vessels,extra-cellular matrix, skin, and umbilical cord. In some embodiments,the cross-linking of HA is through a covalent bond to another molecule,such as a protein. In some embodiments, HA is covalently bound to theheavy chain of inter-α-trypsin inhibitor (IαI). In some embodiments, theratio of protein to HA in the preparation of fetal support tissue isless than about 500:1, less than about 200:1, less than about 100:1,less than about 50:1, or less than about 10:1 protein:HA. In someembodiments, the ratio of HA to protein in the preparation of fetalsupport tissue is less than about 500:1, less than about 200:1, lessthan about 100:1, less than about 50:1, or less than about 10:1HA:protein.

TSG-6 is a hyaluronan binding protein that plays a role in extracellularmatrix remodeling, cell proliferation, and leucocyte migration. TSG-6can form a complex with the serine protease inhibitor inter-α-inhibitor(IαI) and catalyze the transfer of a heavy chain from IαI to HA. PTX-3is Ca²⁺ dependent ligand binding protein that has a pentameric discoidstructure and are present in plasma. TSP-1 (Thrombospondin 1) is ahomotrimeric glycoprotein having a potent anti-angiogenic and otherbiological activities. TSP-1 is secreted into the extracellular matrixby a variety of cell types.

In some embodiments, the preparation of fetal support tissue comprises apurified component selected from HA, IαI, TSG-6, PTX-3, TSP-1,HC-HA/PTX3, or a combination thereof. In some embodiments thepreparation of fetal support tissue comprises reconstituted HC-HA/PTX3.In some embodiments, the preparation of fetal support tissue comprisespurified HC-HA/PTX3. In some embodiments, the preparation of fetalsupport tissue consists of HC-HA/PTX3. In some embodiments, thepreparation of fetal support tissue comprises purified HC-HA/PTX3 at ahigh concentration. In some embodiments, the HC-HA/PTX3 is at aconcentration of 25 to 750 μg/ml, 50 to 500 μg/ml, 50 to 250 μg/ml, orabout 250 ug/ml, about 500 ug/ml, or about 750 ug/ml. In someembodiments, the purified component is obtained from any suitablesource. In some embodiments, the purified component is obtained from afetal support tissue. In some embodiments, the purified component offetal support tissue is obtained from a commercial source. In someembodiments, the purified component of fetal support tissue is isolatedfrom a transgenic organism. In some embodiments, a protein sequence ofthe purified component of fetal support tissue has a similarity of atleast 90%, 93%, 95%, 97%, 99% or 99.5% to a human protein sequence. Insome embodiments, the purified component of fetal support tissue ispurified, substantially purified, partially purified, or are present incrude extracts. In some embodiments, the purified component of fetalsupport tissue is HC-HA/PTX3. In some embodiments, the purifiedcomponent of fetal support tissue is isolated from the preparation offetal support tissue at any time during the process.

In some embodiments, the preparation of fetal support tissue comprisesSmad7. In some embodiments, Smad7 is obtained from any suitable source,such as from amniotic membrane, from a commercial source or isolatedfrom a transgenic organism. In some embodiments, Smad7 is purified,substantially purified, partially purified, or is present in a crudeextract.

In some embodiments, HA, IαI, TSG-6, PTX-3, TSP-1, and optionally Smad7are obtained from the preparation of fetal support tissue. In someembodiments, the preparation of fetal support tissue containing thecombination of HA, IαI, TSG-6, PTX-3, TSP-1 and optionally Smad7 isprepared.

In some embodiments, after homogenization of the fetal support tissue,is centrifuged to remove the insoluble material. In some embodiments,after homogenization of the fetal support tissue, the insoluble materialis left in the preparation of fetal support tissue. In some embodiments,the preparation of fetal support tissue is dried. In some embodiments, apreparation of fetal support tissue is prepared according to a methoddescribed in Example 1.

In some embodiments, the fetal support tissue is obtained from sourcessuch as Bio-Tissue, Inc. (Miami, Fla.) and Baptist Hospital (Miami,Fla.) (under IRB approval). In some embodiments, the fetal supporttissue is obtained in either a fresh, frozen, or previously frozenstate. In some embodiments, the fetal support tissue is washed to removeexcess storage buffer, blood, or contaminants. In some embodiments, theexcess liquid is removed using a brief centrifugation step, or by othermeans. In some embodiments, the fetal support tissue is frozen usingliquid nitrogen or other cooling means to facilitate the subsequenthomogenization. In some embodiments, the source of the fetal supporttissue is a mammal. In some embodiments, the source of the fetal supporttissue is a human. In some embodiments, other sources of fetal supporttissue, such as non-human primate, bovine or porcine, are used.

In some embodiments, the preparation of fetal support tissue is obtainedfrom AM jelly. In some embodiments, the AM jelly is obtained from freshAM tissue. In some embodiments, AM jelly is obtained before freezing thefresh AM tissue. In some embodiments, AM jelly is obtained afterfreezing the fresh AM tissue. In some embodiments, AM jelly is obtainedfrom frozen or previously frozen AM tissue. In some embodiments, the AMjelly is frozen. In some embodiments, the AM jelly is freeze-groundfollowing the procedure for AM preparations as described herein. In someembodiments, the AM jelly is centrifuged. In some embodiments, the AMjelly is lyophilized.

In some embodiments, the preparation of fetal support tissue is madefrom a stroma of the AM. In some embodiments, the stroma is separatedfrom a layer of fresh, frozen, thawed, or otherwise treated AM membrane.In some embodiments, the stroma removal occurs by enzymatic methods,mechanical methods, or by any other suitable means. In some embodiments,the stroma is fresh, frozen, or previously frozen. In some embodiments,the stroma is ground or freeze-ground following the procedure forgenerating the preparation of fetal support tissue from AM as describedherein. In some embodiments, the stroma is centrifuged. In someembodiments, the stroma is lyophilized.

In some embodiment, the preparation is ground fetal support tissue. Insome embodiments, the fetal support tissue is frozen prior to thegrinding process. In some embodiments, the freezing step occurs by anysuitable cooling process. In some embodiments, the fetal support tissueis flash-frozen using liquid nitrogen. In some embodiments, the fetalsupport tissue is placed in an isopropanol/dry ice bath or isflash-frozen in other coolants. In some embodiments, a commerciallyavailable quick freezing process is used. In some embodiments, the fetalsupport tissue is placed in a freezer and allowed to equilibrate to thestorage temperature more slowly, rather than being flash-frozen. In someembodiments, the fetal support tissue is stored at any desiredtemperature. In some embodiments, the fetal support tissue is stored at−20° C. or −80° C.

In some embodiment, the preparation is pulverized fetal support tissue.In some embodiments, the fetal support tissue is pulverized whilefrozen. In some embodiments, fresh, partially thawed, or thawed fetalsupport tissue is used in the grinding step. In some embodiments, thefetal support tissue (fresh, frozen, or thawed) is sliced into pieces ofa desired size with a suitable device, such as a scalpel, then ground tofine particles using a BioPulverizer (Biospec Products, Inc.,Bartlesville, Okla.) or other suitable devices, and homogenized with ahomogenization device such as a Tissue Tearor (Biospec Products, Inc.,Dremel, Wis.), in a suitable solution, forming a homogenate.Non-limiting examples of solutions include, but are not limited to,phosphate buffered saline (PBS), DMEM, NaCl solution, and water. In someembodiments, the pH of the solution is adjusted as needed. In someembodiments, the pH range is from about 5.5 or 6.0 to about 8.5. In someembodiments, the frozen tissue is ground in a solution having a pH ofbetween about 6.3 and about 7.8.

In some embodiment, the preparation is a homogenate of fetal supporttissue. In some embodiments, the homogenate is mixed at any suitablespeed, temperature, or other parameters. In some embodiments, the mixingoccurs at a temperature range of from about 1° C., or 3° C., to about 6°C., 10° C., 15° C., or 20° C. In some embodiments, the mixing occurs atabout 4° C. In some embodiments, the homogenate is mixed, for example,from less than about 1 minute, 10 minutes, or 20 minutes to about 1, 2,3 or more hours.

In some embodiments, the homogenate is centrifuged to remove anyremaining insoluble material and/or cellular debris. In someembodiments, the centrifugation is performed using any suitable range oftime, temperature, protein concentration, buffers, and speed. In someembodiments, the centrifugation occurs at a range of about 1,000, 5,000,or 10,000×g to about 20,000×g. In some embodiments, the centrifugationoccurs at about 15,000×g. In some embodiments, the centrifugation occursfor a duration of from less than 1 minute, 5 minutes, 10 minutes, 20minutes, to about 40 minutes, 60 minutes, 1.5 hours, or more. In someembodiments, the supernatant is collected and stored in aliquots at −80°C. In some embodiments, the total protein is quantitated using anysuitable commercial protein analysis kit, such as a BCA assay (Pierce,Rockford, Ill.). Example 2, Table 1 and FIG. 13 describe the analysis ofAM preparations after low speed or high speed centrifugation.

In some embodiments, for biochemical characterization and purification,the above solutions are supplemented with protease inhibitors. Anexemplary mixture of protease inhibitors is the following: 1 μg/mlaprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 mM PMSF. Insome embodiments, a protease inhibitor is not added to the preparationof fetal support tissue if the preparation of fetal support tissue is tobe added to live cells or tissues.

In some embodiment, the preparation is an extract fetal support tissue.In some embodiments, any suitable buffer or liquid is used to prepare anextract of fetal support tissue. Example 2 examines the use of variousextraction buffers (high salt, low salt, PBS, etc.) on total proteincontent and HA in the extract of fetal support tissue (Table 1). Example2 examined the levels of the specific proteins TSG-6 (FIG. 14), PTX-3(FIG. 18), TSP-1 (FIG. 19), and Smad7 (FIG. 20) using several extractionmethods.

In some embodiments, the preparation of fetal support tissue is testedto confirm the presence of specific components or proteins. In someembodiments, the preparation of fetal support tissue is tested for thepresence of molecules including, but not limited to, HA, IαI, TSG-6,PTX-3, TSP-1, and Smad7. In some embodiments, the preparation of fetalsupport tissue is tested to confirm the absence of pathogens at anypoint during the preparation process.

In some embodiments, the preparation of fetal support tissue is a drypowder. In some embodiments, the dry powder does not requirerefrigeration or freezing during storage to keep the dry powder fromdegrading over time. In some embodiments, the dry powder is stored andreconstituted prior to use. In some embodiments, the dry powder isprepared by preparing the freeze-ground fetal support tissue asdescribed herein, then removing at least a portion of the water in thepreparation of fetal support tissue. In some embodiments, the excesswater is removed from the preparation of fetal support tissue by anysuitable means. In some embodiments, is the excess water is removed byuse of lyophilization. In some embodiments, lyophilizing the preparationof fetal support tissue comprises using a commercially availablelyophilizer or freeze-dryer. In some embodiments, suitable equipment isfound, for example, through Virtis (Gardiner, N.Y.); FTS Systems (StoneRidge, N.Y.); and SpeedVac (Savant Instruments Inc., Farmingdale, N.Y.).In some embodiments, the amount of water that is removed is from about5%, 10%, 20%, 30% to about 60, 70, 80, 90, 95 or 99% or more. In someembodiments, substantially all of the excess water is removed from thepreparation of fetal support tissue. In some embodiments, the dry powderis stored. In some embodiments, the storage temperature varies from lessthan about −196° C., −80° C., −50° C., or −20° C. to more than about 23°C. In some embodiments, the dry powder is characterized (weight, proteincontent, etc.) prior to storage.

In some embodiments, the dry powder is reconstituted in a suitablesolution or buffer prior to use. Non-limiting examples of solutionsinclude, but are not limited to, PBS, DMEM, and BSS. In someembodiments, the pH of the solution is adjusted as needed. In someembodiments, the dry powder is reconstituted with a sufficient volume ofsolution to produce a high concentration of the fetal support tissuereconstituted composition. In some embodiments, the dry powder isreconstituted with a sufficient volume of solution to produce a lowconcentration of the fetal support tissue reconstituted composition.

In some embodiments, the dry powder is reconstituted in a cream,ointment, gel, foam or lotion].

In some embodiments, the preparation of fetal support tissue is used toproduce a phenotypic reversal of AMSCs from myofibroblasts tofibroblasts. In some embodiments, the preparation of fetal supporttissue is used to prevent or slow differentiation of various cell types.In some embodiments, many types of cells are treated with thepreparation of fetal support tissue.

Isolated nHC-HA/PTX3 Complexes

In some embodiments, the compositions include isolated native HC-HA/PTX3complexes (nHC-HA/PTX3).

In some embodiments, the nHC-HA/PTX3 complexes are isolated from anisolated cell. In some embodiments, the nHC-HA/PTX3 complexes areisolated from a cultured cell. In some embodiments, the nHC-HA/PTX3complexes are isolated from a stem cell. In some embodiments, thenHC-HA/PTX3 complexes are isolated from a water soluble fraction of anextract prepared from a tissue, such as umbilical cord or amnioticmembrane. In some embodiments, the water soluble fraction is extractedwith an isotonic salt solution. In some embodiments, the nHC-HA/PTX3complexes are isolated from a water insoluble fraction of an extractprepared from a tissue, such as umbilical cord or amniotic membrane. Insome embodiments, the insoluble fraction is extracted with GnHCl.

In some embodiments, the isolated nHC-HA/PTX3 complex is isolated froman amniotic tissue. In some embodiments, the isolated nHC-HA/PTX3complex is isolated from an amniotic membrane or an umbilical cord. Insome embodiments, the isolated nHC-HA/PTX3 complex is isolated fromfresh, frozen or previously frozen placental amniotic membrane (PAM),fresh, frozen or previously frozen umbilical cord amniotic membrane(UCAM), fresh, frozen or previously frozen placenta, fresh, frozen orpreviously frozen umbilical cord, fresh, frozen or previously frozenchorion, fresh, frozen or previously frozen amnion-chorion, or anycombinations thereof. Such tissues can be obtained from any mammal, suchas, for example, but not limited to a human, non-human primate, cow orpig.

In some embodiments, the nHC-HA/PTX3 is purified by any suitable method.In some embodiments, the nHC-HA/PTX3 complex is purified bycentrifugation (e.g., ultracentrifugation, gradient centrifugation),chromatography (e.g., ion exchange, affinity, size exclusion, andhydroxyapatite chromatography), gel filtration, or differentialsolubility, ethanol precipitation or by any other available techniquefor the purification of proteins (See, e.g., Scopes, ProteinPurification Principles and Practice 2nd Edition, Springer-Verlag, NewYork, 1987; Higgins, S. J. and Hames, B. D. (eds.), Protein Expression:A Practical Approach, Oxford Univ Press, 1999; and Deutscher, M. P.,Simon, M. I., Abelson, J. N. (eds.), Guide to Protein Purification:Methods in Enzymology (Methods in Enzymology Series, Vol 182), AcademicPress, 1997, all incorporated herein by reference).

In some embodiments, the nHC-HA/PTX3 is isolated from an extract. Insome embodiments, the extract is prepared from an amniotic membraneextract. In some embodiments, the extract is prepared from an umbilicalcord extract. In some embodiments, the umbilical cord extract comprisesumbilical cord stroma and/or Wharton's jelly. In some embodiments, thenHC-HA/PTX3 complex is contained in an extract that is prepared byultracentrifugation. In some embodiments, the nHC-HA/PTX3 complex iscontained in an extract that is prepared by ultracentrifugation using aCsCl/4-6M guanidine HCl gradient. In some embodiments, the extract isprepared by at least 2 rounds of ultracentrifugation. In someembodiments, the extract is prepared by more than 2 rounds ofultracentrifugation (i.e. nHC-HA/PTX3 2nd). In some embodiments, theextract is prepared by at least 4 rounds of ultracentrifugation (i.e.nHC-HA/PTX3 4th). In some embodiments, the nHC-HA/PTX3 complex comprisesa small leucine-rich proteoglycan. In some embodiments, the nHC-HA/PTX3complex comprises HC1, HA, PTX3 and/or a small leucine-richproteoglycan.

In some embodiments, ultracentrifugation is performed on an extractprepared by extraction in an isotonic solution. In some embodiments, theisotonic solution is PBS. For example, in some embodiments the tissue ishomogenized in PBS to produce a homogenized sample. The homogenizedsample is then separated into a soluble portion and insoluble portion bycentrifugation. In some embodiments, ultracentrifugation is performed onthe soluble portion of the PBS-extracted tissue. In such embodiments,the nHC-HA/PTX3 purified by ultracentrifugation of the PBS-extractedtissue called an nHC-HA/PTX3 soluble complex. In some embodiments, thenHC-HA soluble complex comprises a small leucine-rich proteoglycan. Insome embodiments, the nHC-HA/PTX3 soluble complex comprises HC1, HA,PTX3 and/or a small leucine-rich proteoglycan.

In some embodiments, ultracentrifugation is performed on an extractprepared by direct guanidine HCl extraction (e.g. 4-6 M GnHCl) of theamniotic membrane and/or umbilical cord tissue. In some embodiments, theGnHCl extract tissues is then centrifuged to produce GnHCl soluble andGnHCl insoluble portions. In some embodiments, ultracentrifugation isperformed on the GnHCl soluble portion. In such embodiments, thenHC-HA/PTX3 purified by ultracentrifugation of the guanidineHCl-extracted tissue is called an nHC-HA/PTX3 insoluble complex. In someembodiments, the nHC-HA insoluble complex comprises a small leucine-richproteoglycan. In some embodiments, the nHC-HA/PTX3 insoluble complexcomprises HC1, HA, PTX3 and/or a small leucine-rich proteoglycan.

In some embodiments, ultracentrifugation is performed on an extractprepared by further guanidine HCl extraction of the insoluble portion ofthe PBS-extracted tissue. For example, in some embodiments the tissue ishomogenized in PBS to produce a homogenized sample. The homogenizedsample is then separated into a soluble portion and insoluble portion bycentrifugation. The insoluble portion is then further extracted inguanidine HCl (e.g. 4-6 M GnHCl) and centrifuged to produce a guanidineHCl soluble and insoluble portions. In some embodiments,ultracentrifugation is performed on the guanidine HCl soluble portion.In such embodiments, the nHC-HA/PTX3 purified by ultracentrifugation ofthe guanidine HCl-extracted tissue is called an nHC-HA/PTX3 insolublecomplex. In some embodiments, the nHC-HA insoluble complex comprises asmall leucine-rich proteoglycan. In some embodiments, the nHC-HA/PTX3insoluble complex comprises HC1, HA, PTX3 and/or a small leucine-richproteoglycan.

In some embodiments, the method of purifying the isolated nHC-HA/PTX3extract comprises: (a) dissolving the isolated extract (e.g. prepared bythe soluble or insoluble method described herein) in CsCl/4-6M guanidineHCl at the initial density of 1.35 g/ml, to generate a CsCl mixture, (b)centrifuging the CsCl mixture at 125,000×g for 48 h at 15° C., togenerate a first purified extract, (c) extracting the first purifiedextract and dialyzing it against distilled water to remove CsCl andguanidine HCl, to generate a dialysate. In some embodiments, the methodof purifying the isolated extract further comprises (d) mixing thedialysate with 3 volumes of 95% (v/v) ethanol containing 1.3% (w/v)potassium acetate at 0° C. for 1 h, to generate a firstdialysate/ethanol mixture, (e) centrifuging the first dialysate/ethanolmixture at 15,000×g, to generate a second purified extract, and (f)extracting the second purified extract. In some embodiments, the methodof purifying the isolated extract further comprises: (g) washing thesecond purified extract with ethanol (e.g., 70% ethanol), to generate asecond purified extract/ethanol mixture; (h) centrifuging the secondpurified extract/ethanol mixture, to generate a third purified extract;and (i) extracting the third purified extract. In some embodiments, themethod of purifying the isolated extract further comprises: (j) washingthe third purified extract with ethanol (e.g., 70% ethanol), to generatea third purified extract/ethanol mixture; (k) centrifuging the thirdpurified extract/ethanol mixture, to generate a forth purified extract;and (l) extracting the forth purified extract. In some embodiments, thepurified extract comprises an nHC-HA/PTX3 complex.

In some embodiments, the nHC-HA/PTX3 complex is purified byimmunoaffinity chromatography. In some embodiments, anti HC1 antibodies,anti-HC2 antibodies, or both are generated and affixed to a stationarysupport. In some embodiments, the unpurified HC-HA complex (i.e., themobile phase) is passed over the support. In certain instances, theHC-HA complex binds to the antibodies (e.g., via interaction of (a) ananti-HC1 antibody and HC1, (b) an anti-HC2 antibody and HC2, (c) ananti-PTX antibody and PTX3, (d) an anti-SLRP antibody and the SLRP, or(e) any combination thereof). In some embodiments the support is washed(e.g., with PBS) to remove any unbound or loosely bound molecules. Insome embodiments, the support is then washed with a solution thatenables elution of the nHC-HA/PTX3 complex from the support (e.g., 1%SDS, 6M guanidine-HCl, or 8M urea).

In some embodiments, the nHC-HA/PTX3 complex is purified by affinitychromatography. In some embodiments, HABP is generated and affixed to astationary support. In some embodiments, the unpurified nHC-HA/PTX3complex (i.e., the mobile phase) is passed over the support. In certaininstances, the nHC-HA/PTX3 complex binds to the HABP. In someembodiments the support is washed (e.g., with PBS) to remove any unboundor loosely bound molecules. In some embodiments, the support is thenwashed with a solution that enables elution of the HC-HA complex fromthe support.

In some embodiments, the nHC-HA/PTX3 complex is purified by acombination of HABP affinity chromatography, and immunoaffinitychromatography using anti HC1 antibodies, anti-HC2 antibodies, anti-PTX3antibodies, antibodies against a SLRP or a combination of SLRPs, or anycombination of antibodies thereof.

In some embodiments, the nHC-HA/PTX3 complex is purified from theinsoluble fraction as described herein using one or more antibodies. Insome embodiments, the nHC-HA/PTX3 complex is purified from the insolublefraction as described herein using anti-SLRP antibodies.

In some embodiments, the nHC-HA/PTX3 complex is purified from thesoluble fraction as described herein. In some embodiments, thenHC-HA/PTX3 complex is purified from the soluble fraction as describedherein using anti-PTX3 antibodies.

In some embodiments, the nHC-HA/PTX3 complex comprises a small leucinerich proteoglycan (SLRP). In some embodiments, the nHC-HA/PTX3 complexcomprises a class I, class II or class II SLRP. In some embodiments, thesmall leucine-rich proteoglycan is selected from among class I SLRPs,such as decorin and biglycan. In some embodiments, the smallleucine-rich proteoglycan is selected from among class II SLRPs, such asfibromodulin, lumican, PRELP (proline arginine rich end leucine-richprotein), keratocan, and osteoadherin. In some embodiments, the smallleucine-rich proteoglycan is selected from among class III SLRPs, suchas epipycan and osteoglycin. In some embodiments, the small leucine-richproteoglycan is selected from among bikunin, decorin, biglycan, andosteoadherin. In some embodiments, the small leucine-rich proteincomprises a glycosaminoglycan. In some embodiments, the smallleucine-rich proteoglycan comprises keratan sulfate.

rcHC-HA/PTX3 Complexes

In some embodiments, the compositions comprise reconstituted HC-HA/PTX3complexes (rcHC-HA/PTX3) with or without SLRPs.

In some embodiments, a method for generating reconstituted HC-HA/PTX3complexes comprises (a) contacting immobilized high molecular weighthyaluronan (HMW HA) with pentraxin 3 (PTX3) under suitable conditions toform a PTX3/HA complex, and (b) contacting the PTX3/HA complex with IαIand Tumor necrosis factor-Stimulated Gene-6 (TSG-6). Provided herein arercHC-HA/PTX3 complexes produced by such method. In some embodiments,TSG-6 catalyzes the transfer of heavy chain 1 (HC1) of inter-α-inhibitor(IαI) to HA. In some embodiments, HC1 of IαI forms a covalent linkagewith HA. In some embodiments, the steps (a) and (b) of the method areperformed sequentially in order.

In some embodiments, a method for generating reconstituted HC-HA/PTX3complexes comprises contacting a PTX3/HA complex with IαI and TSG-6. Insome embodiments, TSG-6 catalyzes the transfer of heavy chain 1 (HC1) ofinter-α-inhibitor (IαI) to HA. Provided herein are rcHC-HA/PTX3complexes produced by such method. In some embodiments, HC1 of IαI formsa covalent linkage with HA.

In some embodiments, a method for generating a complex of HA bound toPTX3 comprises contacting immobilized high molecular weight hyaluronan(HMW HA) with pentraxin 3 (PTX3) under suitable conditions to form aPTX3/HA complex. Provided herein are PTX3/HA complexes produced by suchmethod.

In some embodiments, a method for generating reconstituted HC-HA/PTX3complexes comprises (a) contacting immobilized high molecular weighthyaluronan (HMW HA) with IαI and TSG-6 to HA to form an HC-HA complexpre-bound to TSG-6 and (b) contacting the HC-HA complex with pentraxin 3(PTX3) under suitable conditions to form an rcHC-HA/PTX3 complex.Provided herein are rcHC-HA/PTX3 complexes produced by such method. Insome embodiments, HC1 of IαI forms a covalent linkage with HA. In someembodiments, the steps (a) and (b) of the method are performedsequentially in order. In some embodiments, the method comprisescontacting an HC-HA complex pre-bound to TSG-6 with PTX3.

In some embodiments, the method comprises first contacting highmolecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) undersuitable conditions to form a PTX3/HA complex, then contacting thePTX3/HA complex with IαI and TSG-6.

In some embodiments, the IαI protein and TSG-6 protein are contacted tothe complex at a molar ratio of about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, 10:1, 15:1, or 20:1 (IαI:TSG-6). In some embodiments the ratioof IαI:TSG-6 ranges from about 1:1 to about 20:1, such as about 1:1 toabout 10:1, such as about 1:1 to 5 about:1, such as about 1:1 to about3:1. In some embodiments, the ratio of IαI:TSG-6 is 3:1 or higher. Insome embodiments, the ratio of IαI:TSG-6 is 3:1.

In some embodiments, the steps (a) and (b) of the method are performedsequentially in order. In some embodiments, the method comprisescontacting a PTX3/HA complex with IαI and TSG-6.

In certain instances, TSG-6 interacts with IαI and forms covalentcomplexes with HC1 and HC2 of IαI (i.e. HC1.TSG-6 and HC2.TSG-6). Incertain instances, in the presence of HA, the HCs are transferred to HAto form rcHC-HA. In some embodiments, a TSG-6.HC1 complex is added topre-bound PTX3/HA complex to catalyze the transfer of HC1 to HA. In someembodiments, the method comprises first contacting immobilized highmolecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) undersuitable conditions to form a PTX3/HA complex, then contacting thePTX3/HA complex with a HC1.TSG-6 complex. In some embodiments, acombination of HC1.TSG-6 complex and HC2.TSG-6 complex is added to aPTX3/HA complex.

In some embodiments, the step of contacting PTX3 to immobilized HMW HAoccurs for at least 10 minutes, at least 30 minutes, at least 1 hour, atleast 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, atleast 6 hours, at least 12 hours, or at least 24 hours or longer. Insome embodiments, the step of contacting PTX3 to immobilized HMW HAoccurs for at least 2 hours or longer. In some embodiments, the step ofcontacting PTX3 to immobilized HMW HA occurs for at least 2 hours. Insome embodiments, the step of contacting PTX3 to immobilized HMW HAoccurs at 37° C. In some embodiments, the step of contacting PTX3 toimmobilized HMW HA occurs in 5 mM MgCl₂ in PBS.

In some embodiments, the step of contacting the PTX3/HA complex with IαIand TSG-6 to HA occurs for at least 10 minutes, at least 30 minutes, atleast 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, atleast 5 hours, at least 6 hours, at least 12 hours, or at least 24 hoursor longer. In some embodiments the step of contacting the PTX3/HAcomplex with a HC1.TSG-6 complex and/or a HC2.TSG-6 complex occurs forat least 10 minutes, at least 30 minutes, at least 1 hour, at least 2hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6hours, at least 12 hours, or at least 24 hours or longer. In someembodiments the step of contacting the PTX3/HA complex with a HC1.TSG-6complex and/or a HC2.TSG-6 complex occurs for at least 2 hours orlonger. In some embodiments the step of contacting the PTX3/HA complexwith a HC1.TSG-6 complex and/or a HC2.TSG-6 complex occurs for at least2 hours. In some embodiments the step of contacting the PTX3/HA complexwith a HC1.TSG-6 complex and/or a HC1.TSG-6 complex occurs at 37° C. Insome embodiments the step of contacting the PTX3/HA complex with aHC1.TSG-6 complex and/or a HC1.TSG-6 complex occurs in 5 mM MgCl₂ inPBS.

In some embodiments, the method comprises contacting high molecularweight hyaluronan (HMW HA) with a pentraxin 3 (PTX3) protein,inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1) and Tumornecrosis factor α-stimulated gene 6 (TSG-6) simultaneously undersuitable conditions to form a HC-HA/PTX3 complex. In some embodiments,the contacting the HMW HA with PTX3, IαI and TSG-6 occurs for at least10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, atleast 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, atleast 12 hours, or at least 24 hours or longer. In some embodiments thestep of contacting the HMW HA, PTX3, IαI, and TSG-6 occurs at 37° C. Insome embodiments the step of contacting the HMW HA, PTX3, IαI, and TSG-6occurs in 5 mM MgCl₂ in PBS.

In some embodiments, the method comprises contacting high molecularweight hyaluronan (HMW HA) with a pentraxin 3 (PTX3) protein,inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1) and Tumornecrosis factor α-stimulated gene 6 (TSG-6) sequentially, in any order,under suitable conditions to form a HC-HA/PTX3 complex. In someembodiments, the contacting the HMW HA with PTX3, IαI and TSG-6 occursfor at least 10 minutes, at least 30 minutes, at least 1 hour, at least2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least6 hours, at least 12 hours, or at least 24 hours or longer. In someembodiments the step of contacting the HMW HA, PTX3, IαI, and TSG-6occurs at 37° C. In some embodiments the step of contacting the HMW HA,PTX3, IαI, and TSG-6 occurs in 5 mM MgCl₂ in PBS.

In some embodiments, the methods for production of an rcHC-HA/PTX3complex further comprises addition of one or more small leucine richproteoglycans (SLRPs). In some embodiments, a method for generatingreconstituted HC-HA/PTX3 complexes comprises (a) contacting immobilizedhigh molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) undersuitable conditions to form a PTX3/HA complex, (b) contacting thePTX3/HA complex with IαI and Tumor necrosis factor-Stimulated Gene-6(TSG-6) and (c) contacting the PTX3/HA complex with one or more SLRPS.Provided herein are rcHC-HA/PTX3 complexes produced by such method. Insome embodiments, TSG-6 catalyzes the transfer of heavy chain 1 (HC1) ofinter-α-inhibitor (IαI) to HA. In some embodiments, HC1 of IαI forms acovalent linkage with HA. In some embodiments, the steps (a), (b), and(c) of the method are performed sequentially in order. In someembodiments, the steps (a), (b), and (c) of the method are performedsimultaneously. In some embodiments, the step (a) of the method isperformed and then steps (b) and (c) of the method are performedsequentially in order. In some embodiments, the step (a) of the methodis performed and then steps (b) and (c) of the method are performedsimultaneously.

In some embodiments, a method for generating reconstituted HC-HA/PTX3complexes comprises (a) contacting immobilized high molecular weighthyaluronan (HMW HA) with IαI and TSG-6 to HA to form an HC-HA complexpre-bound to TSG-6, (b) contacting the HC-HA complex with pentraxin 3(PTX3) and (c) contacting the HC-HA complex with one or more SLRPS undersuitable conditions to form an rcHC-HA/PTX3 complex. Provided herein arercHC-HA/PTX3 complexes produced by such method. In some embodiments, HC1of IαI forms a covalent linkage with HA. In some embodiments, the methodcomprises contacting an HC-HA complex pre-bound to TSG-6 with PTX3. Insome embodiments, the steps (a), (b), and (c) of the method areperformed sequentially in order. In some embodiments, the steps (a),(b), and (c) of the method are performed simultaneously. In someembodiments, the step (a) of the method is performed and then steps (b)and (c) of the method are performed sequentially in order. In someembodiments, the step (a) of the method is performed and then steps (b)and (c) of the method are performed simultaneously.

In some embodiments, the SLRP is selected from among a class I, class IIor class II SLRP. In some embodiments, the SLRP is selected from amongclass I SLRPs, such as decorin and biglycan. In some embodiments, thesmall leucine-rich proteoglycan is selected from among class II SLRPs,such as fibromodulin, lumican, PRELP (proline arginine rich endleucine-rich protein), keratocan, and osteoadherin. In some embodiments,the small leucine-rich proteoglycan is selected from among class IIISLRPs, such as epipycan and osteoglycin. In some embodiments, the smallleucine-rich proteoglycan is selected from among bikunin, decorin,biglycan, and osteoadherin. In some embodiments, the small leucine-richprotein comprises a glycosaminoglycan. In some embodiments, the smallleucine-rich proteoglycan comprises keratan sulfate.

PTX3

In some embodiments, PTX3 for use in the methods is isolated from a cellor a plurality of cells (e.g., a tissue extract). Exemplary cellssuitable for the expression of PTX3 include, but are not limited to,animal cells including, but not limited to, mammalian cells, primatecells, human cells, rodent cells, insect cells, bacteria, and yeast, andplant cells, including, but not limited to, algae, angiosperms,gymnosperms, pteridophytes and bryophytes. In some embodiments, PTX3 foruse in the methods is isolated from a human cell. In some embodiments,PTX3 for use in the methods is isolated from a cell that is stimulatedwith one or more proinflammatory cytokines to upregulate PTX3expression. In some embodiments, the proinflammatory cytokine is IL-1 orTNF-α.

In some embodiments, PTX3 for use in the methods is isolated from anamniotic membrane cell. In some embodiments, PTX3 for use in the methodsis isolated from an amniotic membrane cell from an umbilical cord. Insome embodiments, the amniotic membrane cell is stimulated with or moreproinflammatory cytokines to upregulate PTX3 expression. In someembodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, PTX3 for use in the methods is isolated from anumbilical cord cell. In some embodiments, the umbilical cord cell isstimulated with or more proinflammatory cytokines to upregulate PTX3expression. In some embodiments, the proinflammatory cytokine is IL-1 orTNF-α.

In some embodiments, PTX3 for use in the methods is isolated from anamniotic epithelial cell. In some embodiments, PTX3 for use in themethods is isolated from an umbilical cord epithelial cell. In someembodiments, the amniotic epithelial cell or umbilical cord epithelialcell is stimulated with or more proinflammatory cytokines to upregulatePTX3 expression. In some embodiments, the proinflammatory cytokine isIL-1 or TNF-α.

In some embodiments, PTX3 for use in the methods is isolated from anamniotic stromal cell. In some embodiments, PTX3 for use in the methodsis isolated from an umbilical cord stromal cell. In some embodiments,the amniotic stromal cell or umbilical cord stromal cell is stimulatedwith or more proinflammatory cytokines to upregulate PTX3 expression. Insome embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, PTX3 for use in the methods is a native PTX3protein isolated from a cell. In some embodiments, the cell isstimulated with or more proinflammatory cytokines to upregulate PTX3expression. In some embodiments, the proinflammatory cytokine is IL-1 orTNF-α.

In some embodiments, PTX3 is prepared by recombinant technology. In someembodiments, PTX3 is expressed from a recombinant expression vector. Insome embodiments, nucleic acid encoding PTX3 is operably linked to aconstitutive promoter. In some embodiments, nucleic acid encoding PTX3is operably linked to an inducible promoter. In some embodiments, PTX3is expressed in a transgenic animal. In some embodiments, PTX3 is arecombinant protein. In some embodiments, PTX3 is a recombinant proteinisolated from a cell. In some embodiments, PTX3 is a recombinant proteinproduced in a cell-free extract.

In some embodiments, PTX3 is purified from amniotic membrane, umbilicalcord, umbilical cord amniotic membrane, chorionic membrane, amnioticfluid, or a combination thereof. In some embodiments, PTX3 is purifiedfrom amniotic membrane cells. In some embodiments, the amniotic membranecell is an amniotic epithelial cell. In some embodiments, the amnioticmembrane cell is an umbilical cord epithelial cell. In some embodiments,the amniotic membrane cell is an amniotic stromal cell. In someembodiments, the amniotic membrane cell is an umbilical cord stromalcell. In some embodiments, the amniotic membrane cell is stimulated withor more proinflammatory cytokines to upregulate PTX3 expression. In someembodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, PTX3 is not isolated from a cell or a plurality ofcells (e.g., a tissue extract).

In some embodiments, PTX3 comprises a polypeptide having the sequenceset forth in SEQ ID NO: 33 or a variant thereof having at least 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acididentity to the polypeptide having the sequence set forth in SEQ ID NO:33. Exemplary variants include, for example, species variants, allelicvariants and variants that contain conservative and non-conservativeamino acid mutations. In some embodiments, PTX3 comprises a fragment ofPTX3 sufficient to bind to HA and facilitate the formation ofrcHC-HA/PTX3 complex. In some embodiments, PTX3 comprises Glu18 toSer277 of human PTX3. Variants of PTX3 for use in the provided methodsinclude variants with an amino acid modification that is an amino acidreplacement (substitution), deletion or insertion. In some embodiments,such modification improves one or more properties of the PTX3polypeptides such as improving the one or more therapeutic properties ofthe rcHC-HA/PTX3 complex (e.g., anti-inflammatory, anti-immune,anti-angiogenic, anti-scarring, anti-adhesion, regeneration or othertherapeutic activities as described herein).

In some embodiments PTX3 protein is obtained from a commercial source.An exemplary commercial source for PTX3 is, but is not limited to, PTX3(Catalog No. 1826-TS; R&D Systems, Minneapolis, Minn.).

In some embodiments, the PTX3 protein used in the methods is amultimeric protein. In some embodiments, the PTX3 protein used in themethods is a homomultimer. In some embodiments, the homomultimer is adimer, trimer, tetramer, hexamer, pentamer, or octamer. In someembodiments, the PTX3 homomultimer is a trimer, tetramer, or octamer. Inparticular embodiments, the PTX3 homomultimer is an octamer. In someembodiments, the multimerization domain is modified to improvemultimerization of the PTX3 protein. In some embodiments, themultimerization domain is replaced with a heterogeneous multimerizationdomain (e.g., an Fc multimerization domain or leucine zipper) that whenfused to PTX3 improves the multimerization of PTX3.

TSG-6

In some embodiments, TSG-6 for use in the methods is isolated from acell or a plurality of cells (e.g., a tissue extract). Exemplary cellssuitable for the expression of TSG-6 include, but are not limited to,animal cells including, but not limited to, mammalian cells, primatecells, human cells, rodent cells, insect cells, bacteria, and yeast, andplant cells, including, but not limited to, algae, angiosperms,gymnosperms, pteridophytes and bryophytes. In some embodiments, TSG-6for use in the methods is isolated from a human cell. In someembodiments, TSG-6 for use in the methods is isolated from a cell thatis stimulated with one or more proinflammatory cytokines to upregulateTSG-6 expression. In some embodiments, the proinflammatory cytokine isIL-1 or TNF-α.

In some embodiments, TSG-6 for use in the methods is isolated from anamniotic membrane cell. In some embodiments, TSG-6 for use in themethods is isolated from an amniotic membrane cell from an umbilicalcord. In some embodiments, TSG-6 for use in the methods is isolated froman amniotic membrane cell that is stimulated with one or moreproinflammatory cytokines to upregulate TSG-6 expression. In someembodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, TSG-6 for use in the methods is isolated from anumbilical cord cell. In some embodiments, TSG-6 for use in the methodsis isolated from an umbilical cord cell that is stimulated with one ormore proinflammatory cytokines to upregulate TSG-6 expression. In someembodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, TSG-6 for use in the methods is isolated from anamniotic epithelial cell. In some embodiments, TSG-6 for use in themethods is isolated from an umbilical cord epithelial cell. In someembodiments, TSG-6 for use in the methods is isolated from an amnioticepithelial cell or an umbilical cord epithelial cell that is stimulatedwith one or more proinflammatory cytokines to upregulate TSG-6expression. In some embodiments, the proinflammatory cytokine is IL-1 orTNF-α.

In some embodiments, TSG-6 for use in the methods is isolated from anamniotic stromal cell. In some embodiments TSG-6 for use in the methodsis isolated from an umbilical cord stromal cell. In some embodiments,TSG-6 for use in the methods is isolated from an amniotic stromal cellor an umbilical cord stromal cell that is stimulated with one or moreproinflammatory cytokines to upregulate TSG-6 expression. In someembodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, TSG-6 for use in the methods is a native TSG-6protein isolated from a cell. In some embodiments, the cell isstimulated with or more proinflammatory cytokines to upregulate TSG-6expression. In some embodiments, the proinflammatory cytokine is IL-1 orTNF-α.

In some embodiments, TSG-6 is prepared by recombinant technology. Insome embodiments, TSG-6 is expressed from a recombinant expressionvector. In some embodiments, nucleic acid encoding TSG-6 is operablylinked to a constitutive promoter. In some embodiments, nucleic acidencoding TSG-6 is operably linked to an inducible promoter. In someembodiments, TSG-6 is expressed in a transgenic animal. In someembodiments, TSG-6 is a recombinant protein. In some embodiments, TSG-6is a recombinant protein isolated from a cell. In some embodiments,TSG-6 is a recombinant protein produced in a cell-free extract.

In some embodiments, TSG-6 is purified from amniotic membrane, amnioticmembrane, chorionic membrane, amniotic fluid, or a combination thereof.In some embodiments, PTX3 is purified from amniotic membrane cells. Insome embodiments, the amniotic membrane cell is an amniotic epithelialcell. In some embodiments, the amniotic epithelial cell is an umbilicalcord epithelial cell. In some embodiments, the amniotic membrane cell isan amniotic stromal cell. In some embodiments, the amniotic membranecell is an umbilical cord stromal cell. In some embodiments, theamniotic membrane cell is stimulated with or more proinflammatorycytokines to upregulate TSG-6 expression. In some embodiments, theproinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, TSG-6 is not isolated from a cell or a plurality ofcells (e.g., a tissue extract).

In some embodiments, TSG-6 comprises a fragment of TSG-6 that issufficient to facilitate or catalyze the transfer HC1 of IαI to HA. Insome embodiments, TSG-6 comprises the link module of TSG-6. In someembodiments, TSG-6 comprises amino acids Trp18 through Leu277 of TSG-6.In some embodiments, TSG-6 comprises a polypeptide having the sequenceset forth in SEQ ID NO: 2 or a variant thereof having at least 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acididentity to the polypeptide having the sequence set forth in SEQ ID NO:2. Exemplary variants include, for example, species variants, allelicvariants and variants that contain conservative and non-conservativeamino acid mutations. Natural allelic variants of human TSG-6 include,for example, TSG-6 containing the amino acid replacement Q144R. Variantsof TSG-6 or HA binding fragments thereof for use in the provided methodsinclude variants with an amino acid modification that is an amino acidreplacement (substitution), deletion or insertion. In some embodiments,such modification improve one or more properties of the TSG-6polypeptides such as improved transfer of HC1 of IαI to HA or improvedrelease of the TSG-6 polypeptide from the rcHC-HA/PTX3 complex followingtransfer of HC1 of IαI to HA.

In some embodiments, TSG-6 comprises an affinity tag. Exemplary affinitytags include but are not limited to a hemagglutinin tag, apoly-histidine tag, a myc tag, a FLAG tag, a glutathione-S-transferase(GST) tag. Such affinity tags are well known in the art for use inpurification. In some embodiments, such an affinity tag incorporatedinto the TSG-6 polypeptide as a fusion protein or via a chemical linker.In some embodiments, TSG-6 comprises an affinity tag and the unboundTSG-6 is removed from the rcHC-HA/PTX3 complex by affinity purification.

In some embodiments TSG-6 protein is obtained from a commercial source.An exemplary commercial source for TSG-6 is, but is not limited to,TSG-6 (Catalog No. 2104-TS R&D Systems, Minneapolis, Minn.).

IαI

In some embodiments, the IαI comprises an HC1 chain. In someembodiments, the IαI comprises an HC1 and an HC2 chain. In someembodiments, the IαI comprises an HC1 and bikunin. In some embodiments,the IαI comprises an HC1, and HC2 chain and bikunin. In someembodiments, the IαI comprises an HC1, and HC2 chain and bikunin linkedby a chondroitin sulfate chain.

In some embodiments, IαI is isolated from a biological sample. In someembodiments the biological sample is a biological sample from a mammal.In some embodiments, the mammal is a human. In some embodiments, thebiological sample is a blood, serum, plasma, liver, amniotic membrane,chorionic membrane or amniotic fluid sample. In some embodiments, thebiological sample is a blood, serum, or plasma sample. In someembodiments, the biological sample is a blood sample. In someembodiments, the biological sample is a serum sample. In someembodiments, the biological sample is a plasma sample. In someembodiments, the IαI is purified from human blood, plasma or serum. Insome embodiments, IαI is isolated from human serum. In some embodiments,IαI is not isolated from serum. In some embodiments, IαI for use in themethods is produced in an amniotic membrane cell. In some embodiments,IαI for use in the methods is produced in an umbilical cord cell. Insome embodiments, IαI for use in the methods is produced in an amnioticmembrane cell from an umbilical cord. In some embodiments, IαI for usein the methods is produced in an amniotic epithelial cell. In someembodiments, IαI for use in the methods is produced in an umbilical cordepithelial cell. In some embodiments, IαI for use in the methods isproduced in an amniotic stromal cell. In some embodiments, IαI for usein the methods is produced in an umbilical cord stromal cell. In someembodiments, IαI for use in the methods is produced in a hepatic cell.In some embodiments, IαI is prepared by recombinant technology.

In some embodiments, HC1 of IαI is isolated from a biological sample. Insome embodiments the biological sample is a biological sample from amammal. In some embodiments, the mammal is a human. In some embodiments,the biological sample is a blood, serum, plasma, liver, amnioticmembrane, chorionic membrane or amniotic fluid sample. In someembodiments, the biological sample is a blood, serum, or plasma sample.In some embodiments, the biological sample is a blood sample. In someembodiments, the biological sample is a serum sample. In someembodiments, the biological sample is a plasma sample. In someembodiments, the HC1 of IαI is purified from human blood, plasma orserum. In some embodiments, IαI is isolated from human serum. In someembodiments, HC1 of IαI is not purified from serum. In some embodiments,HC1 of IαI is prepared by recombinant technology. In some embodiments,HC1 of IαI is purified from hepatic cells. In some embodiments, HC1 ofIαI is purified from amniotic membrane cells. In some embodiments, HC1of IαI is purified from amniotic epithelial cells or umbilical cordepithelial cells. In some embodiments, HC1 of IαI is purified fromamniotic stromal cells or umbilical cord stromal cells.

In some embodiments, HC1 comprises a polypeptide having the sequence setforth in SEQ ID NO: 47 or a polypeptide having at least 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to thepolypeptide having the sequence set forth in SEQ ID NO: 47.

In some embodiments, HC2 of IαI is isolated from a biological sample. Insome embodiments the biological sample is a biological sample from amammal. In some embodiments, the mammal is a human. In some embodiments,the biological sample is a blood, serum, plasma, liver, amnioticmembrane, chorionic membrane or amniotic fluid sample. In someembodiments, the biological sample is a blood, serum, or plasma sample.In some embodiments, the biological sample is a blood sample. In someembodiments, the biological sample is a serum sample. In someembodiments, the biological sample is a plasma sample. In someembodiments, the HC2 of IαI is purified from human blood, plasma orserum. In some embodiments, HC2 of IαI is isolated from human serum. Insome embodiments, HC2 of IαI is isolated from human serum. In someembodiments, HC2 of IαI is not isolated from blood serum. In someembodiments, HC2 of IαI is prepared by recombinant technology. In someembodiments, HC2 of IαI is purified from hepatic cells. In someembodiments, HC2 of IαI is purified from amniotic membrane cells. Insome embodiments, HC2 of IαI is purified from amniotic epithelial cellsor umbilical cord epithelial cells. In some embodiments, HC2 of IαI ispurified from amniotic stromal cells or umbilical cord stromal cells.

In some embodiments, HC2 comprises a polypeptide having the sequence setforth in SEQ ID NO: 49 or a polypeptide having at least 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to thepolypeptide having the sequence set forth in SEQ ID NO: 49.

In some embodiments, IαI comprises bikunin. In some embodiments, bikunincomprises a polypeptide having the sequence set forth in SEQ ID NO: 53or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% sequence amino acid identity to the polypeptide having thesequence set forth in SEQ ID NO: 53. In some embodiments, IαI comprisesa chondroitin sulfate chain.

HA

In some embodiments, HA is purified from a cell, tissue or a fluidsample. In some embodiments, HA is obtained from a commercial supplier(e.g., Sigma Aldrich or Advanced Medical Optics, Irvine, Calif. (e.g.,Healon)). In some embodiments, HA is obtained from a commercial supplieras a powder. In some embodiments, HA is expressed in a cell. Exemplarycells suitable for the expression of HA include, but are not limited to,animal cells including, but not limited to, mammalian cells, primatecells, human cells, rodent cells, insect cells, bacteria, and yeast, andplant cells, including, but not limited to, algae, angiosperms,gymnosperms, pteridophytes and bryophytes. In some embodiments, HA isexpressed in a human cell. In some embodiments, HA is expressed in atransgenic animal. In some embodiments, HA is obtained from a cell thatexpresses a hyaluronan synthase (e.g., HAS1, HAS2, and HAS3). In someembodiments, the cell contains a recombinant expression vector thatexpresses an HA synthase. In certain instances, an HA synthase lengthenshyaluronan by repeatedly adding glucuronic acid and N-acetylglucosamineto the nascent polysaccharide as it is extruded through the cellmembrane into the extracellular space.

HA for use in the methods is typically high molecular weight (HMW) HA.In some embodiments, the weight average molecular weight of HMW HA isgreater than about 500 kilodaltons (kDa), such as, for example, betweenabout 500 kDa and about 10,000 kDa, between about 800 kDa and about8,500 kDa, between about 1100 kDa and about 5,000 kDa, or between about1400 kDa and about 3,500 kDa. In some embodiments, the weight averagemolecular weight of HMW HA is about 3000 kDa.

Additional Components

In some embodiments, one or more additional components are added togenerate an rcHC-HA/PTX3 complex. In some embodiments, a small leucinerich proteoglycan (SLRP) is added to generate an rcHC-HA/PTX3 complex.In some embodiments, the SLRP is a class I, class II or class II SLRP.In some embodiments, the SLRP is selected from among class I SLRPs, suchas decorin and biglycan. In some embodiments, the SLRP is selected fromamong class II SLRPs, such as fibromodulin, lumican, PRELP (prolinearginine rich end leucine-rich protein), keratocan, and osteoadherin. Insome embodiments, the SLRP is selected from among class III SLRPs, suchas epipycan and osteoglycin. In some embodiments, the SLRP is selectedfrom among bikunin, decorin, biglycan, and osteoadherin. In someembodiments, the SLRP comprises a glycosaminoglycan. In someembodiments, the SLRP comprises keratan sulfate.

HA Immobilization

In some embodiments, HMW HA is immobilized by any suitable method. Insome embodiments, HMW HA is immobilized to a solid support, such asculture dish, bead, a column or other suitable surfaces, such as, forexample, a surface of an implantable medical device or a portion thereofor on a surface that is subsequently connected to or combined with animplantable medical device as described herein. In some embodiments, HMWHA is immobilized directly to the solid support, such a by chemicallinkage. In some embodiments, HMW HA is attached indirectly to the solidsupport via a linker or an intermediary protein. Numerousheterobifunctional cross-linking reagents that are used to form covalentbonds between amino groups and thiol groups and to introduce thiolgroups into proteins, are known to those of skill in this art. In someembodiments, HMW HA is immobilized directly to the solid support viacrosslinking to the solid support. In some embodiments, HMW HA isimmobilized directly to the solid support without crosslinking to thesolid support. In some embodiments, HMW HA is immobilized directly tothe solid support as a coating. In some embodiments, HMW HA isimmobilized to a Covalink™-NH surface. In some embodiments, HMW HA isimmobilized directly to the solid support as a coating. In someembodiments, HMW HA is immobilized to a Covalink™-NH surface for about16 h at 4° C.

In some embodiments, the method comprises immobilizing HMW HA to a solidsurface via direct linkage to a solid support (i.e. without anintermediary protein). In some embodiments, the solid support is washedto remove unbound HMW HA prior to contacting the immobilized HA withPTX3. In some embodiments, the solid support is washed with washes of 8MGnHCl and PBS to remove unbound HMW HA prior to contacting theimmobilized HA with PTX3.

In some embodiments, the method comprises immobilizing HA to a solidsurface via an intermediary protein or a linker. In some embodiments,the linker is a peptide linker. In some embodiments, the intermediaryprotein is an HA binding protein (HABP). In some embodiments, HABP isfirst attached to a solid support (e.g., by cross-linking, chemicallinkage or via a chemical linker). In some embodiments, the solidsupport comprising HABP is then contacted with HA (e.g., HMW HA) toimmobilize HA to the solid support via binding of the HABP to HA. Insome embodiments, the solid support is washed to remove unbound HMW HAprior to contacting the immobilized HMW HA with PTX3. In someembodiments, the solid support is washed with washes of 8M GnHCl and PBSto remove unbound HMW HA prior to contacting the immobilized HA withPTX3.

In some embodiments, the method comprises immobilizing HA to a solidsurface via attachment of a peptide linker to the solid support andattachment HA to the peptide linker. In some embodiments, the peptidelinker comprises a protease cleavage site.

In some embodiments, the method comprises immobilizing HA to a solidsurface via attachment of a cleavable chemical linker, such as, but notlimited to a disulfide chemical linker.

In some embodiments, the HABP selected for use in the methods is an HABPthat is dissociated from HA following formation of the rcHC-HA/PTX3complex. In some embodiments, the HABP non-covalently binds to HA. Insome embodiments, the method further comprises dissociating thercHC-HA/PTX3 complex from HABP using one or more dissociating agents.Dissociating agents for the disruption of non covalent interactions(e.g., guanidine hydrochloride, urea and various detergents, e.g., SDS)are known in the art. In some embodiments the dissociating agent isurea. In some embodiments the dissociating agent is guanidinehydrochloride. In some embodiments, the dissociation agent is about 4Mto about 8M guanidine-HCl. In some embodiments, the dissociation agentis about 4M, about 5M, about 6M, about 7M, about 8M guanidine-HCl. Insome embodiments, the dissociation agent is about 4M to about 8Mguanidine-HCl in PBS at pH 7.5.

In some embodiments, such dissociating agents are employed to dissociatethe rcHC-HA/PTX3 complex from an intermediary HABP. An HABP for use inthe methods typically is selected such that the binding affinity for HAis strong enough to permit assembly of the rcHC-HA/PTX3 complex but isdissociated from the rcHC-HA/PTX3 complex with a suitable dissociationagent. In some embodiments the dissociating agent is guanidinehydrochloride.

Exemplary HABPs for use with the methods provided herein include, butare not limited to, HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican,neurocan, brevican, phosphacan, TSG-6, CD44, stabilin-1, stabilin-2, orportions thereof (e.g., link modules thereof) sufficient to bind HA. Insome embodiments, the HABP comprises a polypeptide having the sequenceset forth in any of SEQ ID NOS: 54-99 or a polypeptide having at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acididentity to the polypeptide having the sequence set forth in any of SEQID NOS: 54-99. In some embodiments, the HABP is versican. In someembodiments, the HABP is a recombinant protein. In some embodiments, theHABP is a recombinant mammalian protein. In some embodiments, the HABPis a recombinant human protein. In some embodiments, the HABP is arecombinant versican protein or a portion thereof sufficient to bind toHA. In some embodiments, the HABP is a recombinant aggrecan protein or aportion thereof sufficient to bind to HA. In some embodiments, the HABPis a native HABP or a portion thereof sufficient to bind to HA. In someembodiments, the native HABP is isolated from mammalian tissue or cells.In some embodiments, the HABP is isolated from bovine nasal cartilage(e.g. HABP from Seikagaku which contains the HA binding domains ofaggrecan and link protein).

In some embodiments, the HABP comprises a link module of HAPLN1, HAPLN2,HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan,TSG-6, CD44, stabilin-1, or stabilin-2. In some embodiments, the HABPcomprising a link module comprises a polypeptide having the sequence setforth in any of link domains of SEQ ID NOS: 54-99 or a polypeptidehaving at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceamino acid identity to the polypeptide having the sequence set forth inany of link domains of SEQ ID NOS: 54-99. In some embodiments, the HABPcomprises a link module of versican. In some embodiments, the HABPcomprising a link module is a recombinant protein. In some embodiments,the HABP comprising a link module of versican is a recombinant protein.

In some embodiments, the or intermediary protein, such as an HABP,contains a proteolytic cleavage sequence that is recognized by and ishydrolyzed by a site specific protease, such as furin, 3C protease,caspase, matrix metalloproteinase or TEV protease. In such embodiments,assembled rcHC-HA/PTX3 complexes are released from the solid support bycontacting the immobilized complexes with a protease that cleaves thespecific cleavage sequence.

In some embodiments, the rcHC-HA/PTX3 complex is purified. In someembodiments, the rcHC-HA/PTX3 complex is purified by any suitable methodor combination of methods. The embodiments described below are notintended to be exclusive, only exemplary.

In some embodiments, the rcHC-HA/PTX3 complex is purified bychromatography (e.g., ion exchange, affinity, size exclusion, andhydroxyapatite chromatography), gel filtration, centrifugation (e.g.,gradient centrifugation), or differential solubility, ethanolprecipitation or by any other available technique for the purificationof proteins.

In some embodiments, the rcHC-HA/PTX3 complex is purified byimmunoaffinity chromatography. In some embodiments antibodies aregenerated against a component of the rcHC-HA/PTX3 complex (e.g.,anti-HC1, anti-PTX, an antibody against one or more SLRPs of thercHC-HA/PTX3 complex, e.g., anti-bikunin, anti-decorin, anti-biglycan,or anti-osteoadherin) and affixed to a solid support. In someembodiments, the unpurified rcHC-HA/PTX3 complex (i.e., the mobilephase) is passed over the support. In certain instances, thercHC-HA/PTX3 complex binds to the antibodies. In some embodiments, thesupport is washed (e.g., with PBS) to remove any unbound or looselybound molecules. In some embodiments, the support is then washed with asolution that enables elution of the rcHC-HA/PTX3 complex from thesupport (e.g., 1% SDS, 6M guanidine-HCl, or 8M urea). In someembodiments, the dissociating agent is removed from the dissociatedrcHC-HA/PTX3 complex. In some embodiments, the dissociating agent isremoved from the dissociated rcHC-HA/PTX3 complex by a method including,but not limited to, ion-exchange chromatography, dialysis, gelfiltration chromatography, ultrafiltration, or diafiltration.

In some embodiments, the rcHC-HA/PTX3 complex is purified by affinitychromatography. In some embodiments, an HABP is employed to bind to thercHC-HA/PTX3 complex for purification of the complex and affixed to astationary support. In some embodiments, the unpurified rcHC-HA/PTX3complex (i.e., the mobile phase) is passed over the support. In certaininstances, the rcHC-HA/PTX3 complex binds to the HABP. In someembodiments the support is washed (e.g., with PBS) to remove any unboundor loosely bound molecules. In some embodiments, the support is thenwashed with a solution (e.g., a dissociating agent) that enables elutionof the rcHC-HA/PTX3 complex from the support. In some embodiments, thedissociating agent is removed from the dissociated rcHC-HA/PTX3 complexby a method including, but not limited to, ion-exchange chromatography,dialysis, gel filtration chromatography, ultrafiltration, ordiafiltration.

In some embodiments, the rcHC-HA/PTX3 complex is purified by acombination of HABP affinity chromatography, and immunoaffinitychromatography using antibodies against one or more components of thercHC-HA/PTX3 complex.

In some embodiments, one or more components of the rcHC-HA/PTX3 complexdisclosed herein comprise an affinity tag (e.g., a fusion protein ofPTX3 or HC1 with an affinity tag). Exemplary affinity tags that areincorporated into one or more components of the rcHC-HA/PTX3 complex insome embodiments include, but are not limited to, a hemagglutinin tag,poly-histidine, a myc tag, a FLAG tag, or glutathione-S-transferasesequence. In some embodiments, the ligand for the affinity tag isaffixed to the solid support. In some embodiments, the unpurifiedrcHC-HA/PTX3 complex is passed over the support. In certain instances,the rcHC-HA/PTX3 complex binds to the ligand. In some embodiments thesupport is washed (e.g., with PBS) to remove any unbound or looselybound molecules. In some embodiments, the support is then washed with asolution that enables elution of an rcHC-HA/PTX3 complex disclosedherein from the support. In some embodiments, the elution agent isremoved from the dissociated rcHC-HA/PTX3 complex by a method including,but not limited to, ion-exchange chromatography, dialysis, gelfiltration chromatography, ultrafiltration, or diafiltration.

In some embodiments, the PTX3, TSG-6, and/or HC1 are conjugated to alabel. A “label” refers to a detectable compound or composition which isconjugated directly or indirectly to a polypeptide so as to generate alabeled polypeptide. In some embodiments, the label is detectable byitself (e.g., radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, catalyzes chemical alteration of a substratecompound composition which is detectable. Non-limiting examples oflabels include fluorogenic moieties, dyes, fluorescent tags, greenfluorescent protein, or luciferase.

Excipients

In some embodiments, the compositions comprise excipients. In someembodiments, the excipient is chosen from the group comprising pHmodifiers, buffers, collagen, HA, antibiotics, surfactants, stabilizers,proteins, and combinations thereof. In some embodiments, excipientcomprises an extracellular matrix (ECM) component. In some embodiments,the ECM component comprises collagen, fibrin, HA, or a combinationthereof.

Collagen is a major structural protein found in the body. It providessupport for tissues, connects tissue to bone, and provides the structureof the body. When the body is in the healing process, collagen plays arole in helping to build a cellular structure. Hyaluronic acid is anatural sugar found in the synovial joint fluid, the vitreous humor ofthe eye, the cartilage, blood vessels, extra-cellular matrix, skin, andumbilical cord. Fibrin is a protein involved in the clotting of blood.

In some embodiments, the preparation of fetal support tissue is mixedwith collagen, fibrin or HA. Collagen, fibrin and HA can be suitabledelivery vehicles, as AM preparations mixed with collagen or HA wereshown to exert a suppressive effect upon TGF 13 promoter activity.Although the preparations of fetal support tissue were mixed withcollagen gel and HA gel in the experiments described herein, in someembodiments, any soluble form (e.g., liquid) of collagen and HA or otherECM components (e.g., fibrin) is used. In some embodiments, thecollagen, fibrin or HA is derived from any suitable source. In someembodiments, the ratio of AM to collagen, fibrin or HA is varied. Insome embodiments, the ratio of AM to collagen, fibrin, or HA is lessthan about 0.001:1, 0.01:1, 0.05:1, or 0.1:1, to about 1:1, 1.5:1, 2:1,5:1, 10:1, 100:1 or 1000:1 or more is used.

In some embodiments, collagen gel is prepared by diluting the stocksolution (4 mg/ml) with 0.1 N acetic acid and by mixing it withappropriate volume ratios of 20× of DMEM or suitable buffer, and 1 NNaOH, as described in Example 1. In some embodiments, the collagen inthe composition is present at a range of from less than about 2 mg/ml tomore than about 4 mg/ml.

In some embodiments, the HA is a high molecular weight (MW) HA. In someembodiments, various dilutions of high MW HA are prepared by dilutingcommercially prepared HA (Healon™ (10 mg HA/nil) (Pharmacia, LaJolla,Calif.) in DMEM or suitable buffer. In some embodiments, dry powder andwater-soluble forms of the preparation of fetal support tissue arediluted in a solution such as PBS, DMEM, or other solutions into thedesired collagen concentration. In some embodiments, the HA in thepreparation of fetal support tissue is present at a range of from lessthan about 2 μg/ml to more than about 129 μg/ml.

Illustrative Preparations

Examples 8 through 15 represent illustrative methods for preparing thepreparations of fetal support tissue described and used herein.

Compositions

In some embodiments, the composition comprising the preparation of fetalsupport tissue is formulated for administration purposes as a non-soliddosage form. In some embodiments, the non-solid dosage form comprisescombining the preparation with a delivery vehicle to create acomposition such as a solution, drop, suspension, paste, spray,ointment, oil, emulsion, aerosol, coated bandage, patch, cream, lotion,gel, and the like. The formulation used will depend upon the particularapplication. Gels are useful for administering the composition becausethey allow better retention of the active ingredient at the site ofintroduction, allowing the active ingredient to exert its effect for alonger period of time before clearance of the active ingredient. In someembodiments, the composition is formulated as extended-release soliddosage forms (including oral dosage forms).

In some embodiments, the composition is formulated in a conventionalmanner using one or more physiologically acceptable carriers includingexcipients and auxiliaries which facilitate processing of preparation offetal support tissue into compositions which are used pharmaceutically.Proper formulation is dependent upon the route of administration chosen.Any of the well-known techniques, carriers, and excipients may be usedas suitable and as understood in the art.

In some embodiments, the composition of fetal support tissue is in aliquid, suspension, a gel, or lyophilized powder, or other forms. Insome embodiments, the composition is injectable. In some embodiments,the composition of fetal support tissue comprises an antimicrobialagent. In some embodiments, the antimicrobial agent is an antibiotic oranti-fungal agent. In some embodiments, the composition of fetal supporttissue comprises an additional substance to stabilize and/or preservethe composition of fetal support tissue. In some embodiments, thecomposition of fetal support tissue is packaged and stored at roomtemperature, −20° C. or −80° C. prior to use.

In certain embodiments, the composition comprises a pharmaceuticallyacceptable diluent, excipient, or carrier. In some embodiments, thecomposition further comprises other active ingredients, as incombination therapy. In some embodiments, the composition comprisesother medicinal or pharmaceutical agents, carriers, adjuvants, such aspreserving, stabilizing, wetting or emulsifying agents, solutionpromoters, and salts for regulating the osmotic pressure, buffers, or acombination thereof. In some embodiments, the composition comprises anadditional therapeutic agent.

In some embodiments, the composition further comprises a chemicalcomponent, such as a carrier, stabilizer, diluent, dispersing agent,suspending agent, thickening agent, excipient, or a combination thereof.In some embodiments, the composition facilitates administration of thepreparation to the individual. In some embodiments, a therapeuticallyeffective amount of the composition of fetal support tissue isadministered as an injectable composition to an individual having adisease, disorder, or condition to be treated. In some embodiments, theindividual is a mammal. In some embodiments, the mammal is a human. Insome embodiments, the therapeutically effective amount varies dependingon the severity of the disease, the age and relative health of theindividual, the potency of the composition used and other factors. Insome embodiments, the composition is used singly or in combination withone or more therapeutic agents as components of mixtures.

Ophthalmic Compositions:

In some embodiments, the ophthalmic compositions comprise a preparationof a fetal support tissue; and a pharmaceutically acceptable diluent,excipient, vehicle, or carrier. In some embodiments, the ophthalmiccompositions consist essentially of substantially isolated HC-HA/PTX3,reconstituted HC-HA/PTX3, or a combination thereof and apharmaceutically acceptable diluent, excipient, vehicle, or carrier. Insome embodiments, the composition is prepared for local delivery to theeye. In some embodiments, the composition is administered systemically,such as intravenously. In some embodiments, the composition isadministered topically to the eye. In some embodiments, the compositionis formulated into a variety of topically administrable ophthalmiccompositions. In some embodiments, the topically administrableophthalmic composition comprises a solution, suspension, gel orointment. In some embodiments, the composition is formulated forinjection into the eye. In some embodiments, the composition isadministered by intravitreal injection into the eye. In someembodiments, the composition is administered by intraocular injection,subretinal injection, intravitreal injection, periocular administration,subconjunctival injections, retrobulbar injections, intracameralinjections (including into the anterior or vitreous chamber), orsub-Tenon's injections. In some embodiments, the composition isadministered by implants, ophthalmic solutions, ophthalmic suspensions,ophthalmic ointments, ocular implants and ocular inserts, intraocularsolutions, use of iontophoresis, incorporation in surgical irrigatingsolutions, and packs (by way of example only, a saturated cotton pledgetinserted in the fornix).

In some embodiments, the composition is a liquid composition where thepreparation of fetal support tissue is present in solution, insuspension or both. In some embodiments, the composition includes a gelformulation. In other embodiments, the liquid composition is aqueous. Insome embodiments, the composition is an ointment.

In some embodiments, the composition is an aqueous composition. In someembodiments, the aqueous composition is an aqueous solution, suspensionor solution/suspension. In some embodiments, the aqueous composition ispresented in the form of eye drops. In some embodiments, a desireddosage is administered via a known number of drops into the eye. Forexample, for a drop volume of 25 μl, administration of 1-6 drops willdeliver 25-150 μl of the composition. In some embodiments, the aqueouscomposition comprises from about 0.01% to about 50% weight/volume of thepreparation of fetal support tissue or purified component. In someembodiments, the aqueous composition comprises from about 0.1% to about20% weight/volume of the preparation of fetal support tissue or purifiedcomponent. In some embodiments, the aqueous composition comprises fromabout 0.2% to about 10% weight/volume of the preparation of fetalsupport tissue or purified component. In some embodiments, the aqueouscomposition comprises from about 0.5% to about 5%, weight/volume of thepreparation of fetal support tissue or purified component. In someembodiments, the aqueous composition has an ophthalmically acceptable pHand osmolality. “Ophthalmically acceptable” with respect to aformulation, composition or ingredient typically means having nopersistent detrimental effect on the treated eye or the functioningthereof, or on the general health of the subject being treated.Transient effects such as minor irritation or a “stinging” sensation arecommon with topical ophthalmic administration of agents and consistentwith the formulation, composition or ingredient in question being“ophthalmically acceptable.”

In some embodiments, the composition is an aqueous composition andcomprises a polymer as a suspending agent. In some embodiments, theaqueous composition comprises more than one polymer as the suspendingagent. In some embodiments, the polymer comprises a water-solublepolymer, a water-insoluble polymer, or a combination thereof. In someembodiments, the water-soluble polymer comprises a cellulosic polymer.In some embodiments, the cellulosic polymer comprises hydroxypropylmethylcellulose. In some embodiments, the water-insoluble polymercomprises a cross-linked carboxyl-containing polymer. In someembodiments, the aqueous composition comprises an ophthalmicallyacceptable mucoadhesive polymer. In some embodiments, the mucoadhesivepolymer comprises carboxymethylcellulose, carbomer (acrylic acidpolymer), poly (methylmethacrylate), polyacrylamide, polycarbophil,acrylic acid/butyl acrylate copolymer, sodium alginate, dextran, or acombination thereof.

In some embodiments, the composition comprises an ophthalmicallyacceptable solubilizing agent to aid in the solubility of thepreparation of fetal support tissue in the composition. In someembodiments, the composition comprises an ophthalmically acceptablesolubilizing agent to aid in the solubility of purified HC-HA/PTX3 inthe composition. The term “solubilizing agent” generally includes agentsthat result in formation of a micellar solution or a true solution ofthe agent. In some embodiments, the ophthalmically acceptablesolubilizing agent is a nonionic surfactants. In some embodiments, thenonionic surfactant comprises polysorbate 80, glycol, polyglycol,polyethylene glycol 400, glycol ethers, derivatives thereof, or anycombination thereof.

In some embodiments, the composition comprises one or moreophthalmically acceptable pH adjusting agents or buffering agents. Insome embodiments, the pH adjusting agent comprises an acid. In someembodiments, the acid is chosen from a list comprising: acetic, boric,citric, lactic, phosphoric acid, and hydrochloric acid. In someembodiments, the pH adjusting agent comprises a base. In someembodiments, the base is chosen from a list comprising: sodiumhydroxide, sodium phosphate, sodium borate, sodium citrate, sodiumacetate, sodium lactate and tris-hydroxymethylaminomethane. In someembodiments, the buffering agent is chosen from a list comprising:citrate/dextrose, sodium bicarbonate, and ammonium chloride. In someembodiments, the acid, the base or the buffers are included in an amountrequired to maintain pH of the composition in an ophthalmicallyacceptable range.

In some embodiments, the composition comprises an ophthalmicallyacceptable salt in an amount required to bring osmolality of thecomposition into an ophthalmically acceptable range. In someembodiments, the salt comprises sodium, potassium or ammonium cationsand chloride, citrate, ascorbate, borate, phosphate, bicarbonate,sulfate, thiosulfate or bisulfite anions. In some embodiments, the saltis chosen from a list comprising: sodium chloride, potassium chloride,sodium thiosulfate, sodium bisulfite, ammonium sulfate, or a combinationthereof.

In some embodiments, the composition comprises an ophthalmicallyacceptable preservative to inhibit microbial activity. In someembodiments, the preservative comprises a mercury-containing substance,stabilized chlorine dioxide, a quaternary ammonium compound, or acombination thereof. In some embodiments, the mercury-containingsubstance comprises merfen, thiomersal, or a combination thereof. Insome embodiments, the quaternary ammonium compound comprisesbenzalkonium chloride, cetyltrimethylammonium bromide, cetylpyridiniumchloride, or a combination thereof.

In some embodiments, the composition comprises one or moreophthalmically acceptable surfactants to enhance physical stability orfor other purposes. In some embodiments, the surfactant comprises anonionic surfactant. In some embodiments, the nonionic surfactant ischosen from a list comprising: polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40.

In some embodiments, the composition comprises one or more antioxidantsto enhance chemical stability where required. In some embodiments, theantioxidant comprises ascorbic acid, sodium metabisulfite, or acombination thereof.

In some embodiments, the composition is packaged in single-dosenon-reclosable containers. In some embodiments, the composition ispackaged in a multiple-dose reclosable container. In some embodiments,the composition further comprises a preservative when packaged in themultiple-dose reclosable container.

In some embodiments, the composition is in the form of a solid articlethat is inserted between the eye and eyelid or in the conjunctival sac,where it releases the preparation. In some embodiments, the preparationis released to the lacrimal fluid that bathes the surface of the cornea,or directly to the cornea itself, with which the solid article isgenerally in intimate contact. In some embodiments, the solid articlesuitable for implantation in the eye comprises polymers. In someembodiments, the solid article suitable for implantation in the eye isbiodegradable or non-biodegradable.

Injectable Compositions:

In some embodiments, the composition is an injectable composition. Insome embodiments, the injectable compositions comprise a preparation ofa fetal support tissue; and a pharmaceutically acceptable diluent,excipient, vehicle, or carrier. In some embodiments, the injectablecompositions consist essentially of substantially isolated HC-HA/PTX3,reconstituted HC-HA/PTX3, or a combination thereof; and apharmaceutically acceptable diluent, excipient, vehicle, or carrier. Insome embodiments, the injectable composition is suitable forintraocular, intramuscular, subcutaneous, or intravenous injection. Insome embodiments, the injectable composition comprises physiologicallyacceptable sterile aqueous or non-aqueous solutions, dispersions,suspensions or emulsions, and sterile powders for reconstitution intosterile injectable solutions or dispersions. Non-limiting examples ofsuitable aqueous and non-aqueous carriers, diluents, solvents, orvehicles including water, ethanol, polyols (propyleneglycol,polyethylene-glycol, glycerol, cremophor and the like), suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. In some embodiments, properfluidity is maintained by the use of a coating, a surfactant, or acombination thereof. In some embodiments, the coating is lecithin. Insome embodiments, the injectable composition comprises an additive. Insome embodiments, the additive is chosen from the list comprising: apreserving agent, a wetting agent, an emulsifying agent, a dispensingagent, or a combination thereof. In some embodiments, the injectablecomposition comprises an antibacterial or antifungal agent. In someembodiments, the antibacterial or antifungal agent is comprises aparaben, chlorobutanol, phenol, sorbic acid, or a combination thereof.In some embodiments, the injectable composition comprises an isotonicagent. In some embodiments, the isotonic agent comprises sugar, sodiumchloride, or a combination thereof. In some embodiments, the injectablecomposition comprises an absorption delaying agent. In some embodiments,the absorption delaying agent comprises aluminum monostearate gelatin,or a combination thereof.

In some embodiments, the injectable composition is administeredintravenously. In some embodiments, the injectable composition isformulated in an aqueous solution, in a physiologically compatiblebuffer such as Hank's solution, Ringer's solution, a physiologicalsaline buffer, or another suitable solution. In some embodiments, fortransmucosal administration, a penetrant appropriate to the barrier tobe permeated is used in the formulation. Such penetrants are generallyknown in the art. In some embodiments, for a parenteral injection, anappropriate formulation includes aqueous or nonaqueous solutions,preferably with physiologically compatible buffers or excipients. Suchexcipients are generally known in the art.

In some embodiments, parenteral injections involve bolus injection orcontinuous infusion. In some embodiments, the composition is presentedin unit dosage form, e.g., in ampoules or in multi dose containers, withan added preservative. In some embodiments, the injectable compositionis in a formulation suitable for parenteral injection as a sterilesuspensions, solutions or emulsions in oily or aqueous vehicles. In someembodiments, the injectable composition comprises a formulary agent. Insome embodiments, the formulary agent is a suspending agent, stabilizingagent, dispersing agent, or a combination thereof. In some embodiments,the injectable composition for parenteral administration comprises theaqueous solution of preparation of fetal support tissue in water solubleform. In some embodiments, the suspension of the active compounds isprepared as an oily injection suspension. In some embodiments, theinjectable composition comprises a lipophilic solvent or vehicle.Non-limiting examples of lipophilic solvents or vehicles include, butare not limited to, fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes. Insome embodiments, the injectable injection composition contains asubstance which increases the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. In someembodiments, the injectable composition contains suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions. In some embodiments, thepreparation of fetal support tissue is in powder form for constitutionwith a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Methods of Dosing and Treatments Regimens:

In some embodiments, the composition is administered by any suitabletechnique. In some embodiments, composition is administered directly toa target site (e.g., ocular surface, vitreous, etc.). In someembodiments, the composition is administered topically. In someembodiments, the composition is administered parentally (e.g.,subcutaneous). In some embodiments, composition is administeredintraocularly.

In some embodiments, the composition is administered for prophylacticand/or therapeutic applications. In some embodiments, the composition isadministered to an individual already suffering from a disease orcondition, in an amount sufficient to cure or at least partially arrestthe symptoms of the disease or condition. In some embodiments, amountseffective for this use depend on the severity and course of the diseaseor condition, previous therapy, the individual's health status, weight,and response to the drugs, and the judgment of the treating physician.

In some embodiments, the composition is administered to an individualsusceptible to or otherwise at risk of a particular disease, disorder orcondition. Such an amount is defined to be a “prophylactically effectiveamount or dose.” In this use, the precise amounts also depend on theindividual's state of health, weight, and the like. In some embodiments,a dose escalation trial is used to determine a prophylacticallyeffective amount. In some embodiments, any suitable method is used todetermine the prophylactically effective amount. In some embodiments,the prophylactically effective amount depends on the severity and courseof the disease, disorder or condition, previous therapy, theindividual's health status and response to the drugs, and the judgmentof the treating physician.

In the case wherein the individual's condition does not improve, uponthe doctor's discretion the composition is administered chronically,that is, for an extended period of time, including throughout theduration of the individual's life in order to ameliorate or otherwisecontrol or limit the symptoms of the individual's disease or condition.

In the case wherein the individual's status does improve, upon thedoctor's discretion the composition is given continuously or the dose ofdrug being administered is temporarily reduced or temporarily suspendedfor a certain length of time (i.e., a “drug holiday”). In someembodiments, the length of the drug holiday varies between 2 days and 1year, including by way of example only, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250days, 280 days, 300 days, 320 days, 350 days, or 365 days. In someembodiments, the dose reduction during a drug holiday is from 10%-100%,including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the individual's conditions has occurred, amaintenance dose is administered if necessary. Subsequently, the dosageor the frequency of administration, or both, is reduced, as a functionof the symptoms, to a level at which the improved disease, disorder orcondition is retained. In some embodiments, the individual requiresintermittent treatment on a long-term basis upon any recurrence ofsymptoms.

In some embodiments, the amount of the composition administered to theindividual varies depending upon factors such as the disease orcondition and its severity, the identity (e.g., weight, gender, age,overall health) of the individual in need of treatment. In someembodiments, the amount of composition administered is determined in amanner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific preparation,composition, or formulation being administered, the route ofadministration, the condition being treated, and the individual beingtreated. In some embodiments, the amounts or doses employed for adulthuman treatment are in the range of 0.02-5000 mg per day, preferably1-1500 mg per day. In some embodiments, a desired dose is presented in asingle dose or as divided doses administered simultaneously (or over ashort period of time) or at appropriate intervals, for example as two,three, four or more sub-doses per day.

In some embodiments, the composition is in unit dosage forms suitablefor single administration of precise amounts or dosages. In unit dosageform, the composition is divided into unit doses containing appropriateamounts or doses of the composition. In some embodiments, the unitdosage is in the form of a package containing discrete quantities of thecomposition. Non-limiting examples are powders packaged in vials orampoules. In some embodiments, compositions are packaged in single-dosenon-reclosable containers. In some embodiments, multiple-dose reclosablecontainers are used, in which case it is typical to include apreservative in the composition. In some embodiments, the compositionfor parenteral injection are presented in unit dosage form, whichinclude, but are not limited to ampoules, or in multi-dose containers,with an added preservative.

In some embodiments, the daily dosage appropriate for the composition isfrom about 0.01 to 2.5 mg/kg per body weight. An indicated daily dosageis in the range from about 0.5 mg to about 100 mg, convenientlyadministered in divided doses, including, but not limited to, up to fourtimes a day or in extended release form. The foregoing ranges are merelysuggestive, as the number of variables in regard to an individualtreatment regime is large, and considerable excursions from theserecommended values are not uncommon. In some embodiments, the dosage isaltered depending on a number of variables, not limited to the activityof the composition, the disease or condition to be treated, the mode ofadministration, the requirements of the individual, the severity of thedisease or condition being treated, and the judgment of thepractitioner.

In some embodiments, the toxicity and therapeutic efficacy of suchtherapeutic regimens is determined by standard pharmaceutical proceduresin cell cultures or experimental animals, including, but not limited to,the determination of the LD₅₀ (the dose lethal to 50% of the population)and the ED₅₀ (the dose therapeutically effective in 50% of thepopulation). In some embodiments, the dose ratio between the toxic andtherapeutic effects is the therapeutic index and is expressed as theratio between LD₅₀ and ED₅₀. Compositions exhibiting high therapeuticindices are preferred. In some embodiments, data obtained from a cellculture assay or animal study is used in formulating a range of dosagefor use in the individual. In some embodiments, the dosage of thecomposition is within a range of circulating concentrations that includethe ED₅₀ with minimal toxicity. In some embodiments, the dosage varieswithin this range depending upon the dosage form employed and the routeof administration utilized.

Combination Treatments:

In some embodiments, the composition is co-administered with anadditional therapeutic compound. In some embodiments, the additionaltherapeutic agent is not administered in the same composition. In someembodiments, the additional therapeutic agent is administered by adifferent route than the composition. The determination of the mode ofadministration and the advisability of administration, where possible,in the same composition, is well within the knowledge of the skilledclinician. In some embodiments, the initial administration is madeaccording to established protocols known in the art, and then modifiedby the skilled clinician based upon the observed effects, the dosage,modes of administration and times of administration.

In some embodiments, the particular choice of the additional therapeuticcompound used depends upon the diagnosis of the attending physicians andtheir judgment of the condition of the individual and the appropriatetreatment protocol. In some embodiments, the additional therapeuticcompound is administered concurrently (e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) or sequentially,depending upon the nature of the disease, disorder, or condition, thecondition of the individual, and the actual choice of compounds used.The determination of the order of administration, and the number ofrepetitions of administration of each therapeutic agent during atreatment protocol, is well within the knowledge of the skilledphysician after evaluation of the disease being treated and thecondition of the individual.

In some embodiments, a therapeutically-effective dosage varies when thecomposition is used in a combination treatment. In some embodiments, anysuitable method is used to determine a therapeutically effective dosageof a drug and other agents for use in the combination treatmentregimens. In some embodiments, metronomic dosing (i.e., providing morefrequent, lower doses in order to minimize toxic side effects) is usedto determine a therapeutically effective dosage of a drug and otheragents for use in the combination treatment. In some embodiments, thecombination treatment comprises periodic treatments that start and stopat various times to assist with the clinical management of theindividual.

In some embodiments, dosage of the additional therapeutic agent variesdepending on the type of co-drug employed, on the specific drugemployed, on the disease or condition being treated and so forth. Insome embodiments, when co-administered with one or more additionaltherapeutic agents, the composition is administered eithersimultaneously with the additional therapeutic agent, or sequentially.If administered sequentially, the attending physician will decide on theappropriate sequence of administering the composition in combinationwith the additional therapeutic agent.

In some embodiments, multiple additional therapeutic agents areadministered in combination with the composition. In some embodiments,the multiple additional therapeutic agents are administered in any orderor even simultaneously. If simultaneously, the multiple additionaltherapeutic agents are provided in a single, unified form, or inmultiple forms (by way of example only, either as a single pill or astwo separate pills). In some embodiments, one of the additionaltherapeutic agents is given in multiple doses, or both may be given asmultiple doses. In some embodiments, if administration is notsimultaneous, the timing between the multiple doses varies from morethan zero weeks to less than four weeks.

In some embodiments, the dosage regimen to treat, prevent, or amelioratethe condition(s) for which relief is sought, is modified in accordancewith a variety of factors. In some embodiments, the factors comprise: adisorder from which the individual suffers, as well as the age, weight,sex, diet, and medical condition of the individual, or a combinationthereof. In some embodiments, the dosage regimen varies widely anddeviates from the dosage regimens set forth herein.

In some embodiments, the composition and additional therapeutic agentwhich make up the combination therapy are a combined dosage form or inseparate dosage forms intended for substantially simultaneousadministration. In some embodiments, the composition and additionaltherapeutic agent that make up the combination therapy are administeredsequentially, with either the composition or the additional therapeuticagent being administered by a regimen calling for two-stepadministration. In some embodiments, the two-step administration regimencalls for sequential administration of the composition and additionaltherapeutic agent or spaced-apart administration of the composition andadditional therapeutic agent. In some embodiments, the time periodbetween the multiple administration steps ranges from, a few minutes toseveral hours, depending upon the properties of each pharmaceuticalagent, such as potency, solubility, bioavailability, plasma half-lifeand kinetic profile of the composition or additional therapeutic agent.In some embodiments, circadian variation of the composition ortherapeutic agent concentration determines the optimal dose interval.

In some embodiments, the composition is used in combination withprocedures that may provide additional or synergistic benefit to theindividual. By way of example only, individuals are expected to findtherapeutic and/or prophylactic benefit in the methods described herein,wherein the composition or the composition in combination with theadditional therapeutic agent is combined with genetic testing todetermine whether that individual is a carrier of a mutant gene that isknown to be correlated with certain diseases or conditions.

In some embodiments, the composition and combination therapies areadministered before, during or after the occurrence of a disease orcondition, and the timing of administering the composition containing acompound varies. In some embodiments, the composition is used as aprophylactic and administered continuously to individuals with apropensity to develop conditions or diseases in order to prevent theoccurrence of the disease or condition. In some embodiments, thecomposition is administered to the individual during or as soon aspossible after the onset of the symptoms. In some embodiments, theadministration of the composition is initiated within the first 48 hoursof the onset of the symptoms, preferably within the first 48 hours ofthe onset of the symptoms, more preferably within the first 6 hours ofthe onset of the symptoms, and most preferably within 3 hours of theonset of the symptoms. In some embodiments, the initial administrationis via any route practical, such as, for example, an intravenousinjection, a bolus injection, infusion over 5 minutes to about 5 hours,and the like, or combination thereof. In some embodiments, thecomposition is administered as soon as is practicable after the onset ofa disease or condition is detected or suspected, and for a length oftime necessary for the treatment of the disease, such as, for example,from about 1 month to about 3 months. In some embodiments, the length oftreatment varies for each individual, and the length is determined usingthe known criteria. In some embodiments, the composition is administeredfor at least 2 weeks, preferably about 1 month to about 5 years, andmore preferably from about 1 month to about 3 years.

Methods of Treatment:

Disclosed herein, in certain embodiments, are methods for preventing orreducing proliferation, cell migration, and/or EMT of epithelial cellsin an individual in need thereof, comprising administering to theindividual a therapeutically effective amount of an injectablecomposition, comprising: (a) a preparation of a fetal support tissue;and (b) a pharmaceutically acceptable diluent, excipient, vehicle, orcarrier, thereby preventing or reducing the proliferation, cellmigration, and/or EMT of epithelial cells. In some embodiments, the EMTis associated with a disease other than PVR.

Disclosed herein, in certain embodiments, are methods for treating orpreventing of Proliferative Vitreoretinopathy (PVR) in an individual inneed thereof, comprising administering to the individual atherapeutically effective amount of an injectable composition,comprising: (a) a preparation of fetal support tissue; and (b) apharmaceutically acceptable diluent, excipient, vehicle, or carrier,thereby treating or preventing PVR.

In some embodiments, the preparation of fetal support tissue comprisesHC-HA/PTX3. In some embodiments, the preparation of fetal support tissuecomprises purified HC-HA/PTX3. In some embodiments, the preparation offetal support tissue comprises ultracentrifuged HC-HA/PTX3. In someembodiments, the preparation of fetal support tissue consists ofpurified HC-HA/PTX3. In some embodiments, the preparation of fetalsupport tissue comprises reconstituted HC-HA/PTX3. In some embodiments,the preparation of fetal support tissue comprises: high molecular weighthyaluronan (HA) that is cross-linked by a covalent bond to the heavychain of inter-α-trypsin inhibitor (IαI), the high molecular weight HAhaving a molecular weight greater than 1000 kDa. In some embodiments,the preparation comprises: pentraxin 3 (PTX-3). In some embodiments, thepreparation of fetal support tissue comprises: tumor necrosisfactor-stimulated gene 6 protein (TSG-6). In some embodiments, thepreparation of fetal support tissue comprises: thrombospondin-1 (TSP-1).In some embodiments, the ratio of total protein to HA in the compositionis less than 500 parts protein:1 part HA. In some embodiments, the ratioof HA to total protein in the compositions is less than 500 parts HA:1part protein.

In some embodiments, the epithelial cells are human epithelial cells. Insome embodiments, the human epithelial cells are retinal pigmentepithelial cells (RPE). In some embodiments, the human epithelial cellsare renal epithelial cells. In some embodiments, the human epithelialcells are corneal epithelial cells. In some embodiments, the humanepithelial cells are limbal epithelial cells. In some embodiments, thehuman epithelial cells are conjunctival epithelial cells.

In some embodiments, the composition prevents the proliferation and EMTof epithelial cells by inhibiting or suppressing the activity of growthfactors or cytokines. In some embodiments, the growth factors andcytokines are selected from the group consisting of: EGF, FGF-2, PDGF-A,PDGF-AB, PDGF-B, PDGF-C, TGF-β1, TGF-β2, TGF-β3, CTGF, HGF, IGF-1,G-CSF, IL-6, MCP-1, TNF-α, VEGF and IFN-γ. In some embodiments, thecomposition inhibits signaling pathways in epithelial cells to inhibitproliferation and EMT. In some embodiments, the signaling pathways arecanonical Wnt signaling and TGF-β-induced Smad/ZEB signaling.

In some embodiments, the compositions comprise a preparation of fetalsupport tissue prepared from placental tissue, umbilical cord tissue,umbilical cord amniotic membrane tissue, placental amniotic membranetissue, amniotic stromal tissue, amnion-chorion tissue, chorion tissue,amniotic fluid, or combinations thereof. In some embodiments, theplacental tissue, umbilical cord tissue, amniotic membrane tissue,chorion tissue or combinations thereof is homogenized, pulverized orground. In some embodiments, the placental tissue, umbilical cordtissue, amniotic membrane tissue, chorion tissue or combinations thereofis fresh, frozen or has been previously frozen. In some embodiments, acomposition comprises the preparation of fetal support tissue and apharmaceutically acceptable diluent, excipient, or carrier. In someembodiments, the composition further comprises an aqueous adjuvant. Insome embodiments, the composition is for local administration. In someembodiments, the composition is for injection. In some embodiments, thecomposition is formulated for intraocular injection, subretinalinjection, intravitreal injection, periocular injection, subconjunctivalinjection, retrobulbar injection, intracameral injection or sub-Tenon'sinjection.

The methods disclosed herein have many uses including research andclinical applications. In some embodiments, the methods are applied totissues or cells to achieve a desired modulation of physiology. In someembodiments, the methods are used on cell cultures or tissue cultures toachieve a desired effect.

In some embodiments, the methods are used to prevent, lessen, or treatapoptosis in tissues. In some embodiments, the methods are used todecrease or prevent apoptosis in a tissue that has been injured. In someembodiments, the methods are used to prolong the life of organs beingstored prior to transplant. In some embodiments, the methods are used totreat or prevent damage during and after surgical procedures.

In some embodiments, methods are useful for preserving tissues (e.g.,cornea) before transplantation. In some embodiments, the methods lessencellular damage due to the storage process. In some embodiments, themethods are used to decrease the amount of degradation that occurs in atissue that is being stored prior to transplantation or surgicalprocedures. In some embodiments, the preparation or composition is addedto the storage medium, with or without collagen and/or HA. Storedtissues such as eyes, organs, skin, and the like can benefit from thedecreased cellular apoptosis that occurs when the composition is added.

In some embodiments, the methods further comprise storing a donor tissuein a storage medium until transplantation after the donor tissue isharvested. In some embodiments, the composition is added to the storagemedium to prevent cellular apoptosis. In some embodiments, thecomposition is added to storage media for preserving limbal epithelialstem cells. In some embodiments, the composition is added to cellculture medium or digestion medium to prevent cellular (e.g.,keratocyte) apoptosis. Because studies described herein show thatincubation of composition during dispase digestion (a treatment whichmimics surgical and pathological insults such as excimer ablation in PRKand recurrent corneal erosion, respectively) significantly reducedapoptosis of both epithelial cells and keratocytes. In some embodiments,the composition is administered to an eye receiving mechanical scrapingor excimer laser photoablation to attempt to reduce keratocyteapoptosis, and hence reduce corneal haze. In some embodiments, themethods are used in a surgical condition or disease such as recurrentcorneal erosion or keratoconus where the basement membrane is dissolvedto reduce the keratocyte apoptosis.

In some embodiments, the method is used to produce a phenotypic reversalof AMSCs from myofibroblasts to fibroblasts. In some embodiments, themethod is used to prevent or slow differentiation of various cell types.In some embodiments, many types of cells are treated with the method.This method is particularly useful for expanding cell cultures withoutcausing differentiation of the culture to unwanted cell types.

Kits/Articles of Manufacture:

For use in the methods described herein, kits and articles ofmanufacture are also described herein. In some embodiments, the kitscomprise a carrier, package, or container that is compartmentalized toreceive one or more containers such as vials, tubes, and the like, eachof the container(s) including one of the separate elements to be used ina method described herein. In some embodiments, the container is abottle, vial, syringe, or test tube. In some embodiments, the containeris formed from a variety of materials such as glass or plastic. In someembodiments, the kit comprising one or more prefilled syringescomprising a composition disclosed herein.

In some embodiments, the article of manufacture contains packagingmaterials. Packaging materials for use in packaging pharmaceuticalproducts are well known to those of skill in the art. Non-limitingexamples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials,containers, syringes, bottles, and any packaging material suitable for aselected formulation and intended mode of administration and treatment.A wide array of formulations of the preparations and compositionsprovided herein are contemplated as are a variety of treatments for anydisease, disorder, or condition.

In some embodiments, the container includes one or more preparations offetal support tissue, optionally in a composition or in combination withanother agent as disclosed herein. In some embodiments, the containercomprises a sterile access port. In some embodiments, the container isan intravenous solution bag or a vial. In some embodiments, the sterileaccess port is a stopper pierceable by a hypodermic injection needle. Insome embodiments, the kit comprises a composition with an identifyingdescription or label or instructions relating to its use in the methodsdescribed herein.

In some embodiments, the kit comprises one or more additionalcontainers, each with one or more of various materials (such asreagents, optionally in concentrated form, and/or devices) desirablefrom a commercial and user standpoint for use of the compositioncomprising fetal support tissue. Non-limiting examples of such materialsinclude, but not limited to, buffers, diluents, filters, needles,syringes; carrier, package, container, vial and/or tube labels listingcontents and/or instructions for use, and package inserts withinstructions for use. In some embodiments, a set of instructions isincluded.

In some embodiments, a label is on or associated with the container. Insome embodiments, the label is on a container when letters, numbers orother characters forming the label are attached, molded or etched intothe container itself. In some embodiments, the label is associated witha container when it is present within a receptacle or carrier that alsoholds the container, e.g., as a package insert. In some embodiments, thelabel is used to indicate that the contents are to be used for aspecific therapeutic application. In some embodiments, the labelindicates directions for use of the contents, such as in the methodsdescribed herein.

In certain embodiments, the injectable composition is presented in apack or dispenser device which contains one or more unit dosage formscontaining the injectable composition provided herein. In someembodiments, the pack contains metal or plastic foil, such as a blisterpack. In some embodiments, the pack or dispenser device is accompaniedby instructions for administration. In some embodiments, the pack ordispenser device is accompanied with a notice associated with thecontainer in form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the drug for human orveterinary administration. In some embodiments, the notice is thelabeling approved by the U.S. Food and Drug Administration forprescription drugs, or the approved product insert. In some embodiments,the injectable composition is prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition.

The compositions and methods described herein are provided in furtherdetail in the following examples. These examples are provided by way ofillustration and are not intended to limit the invention in any way.

EXAMPLES Example 1: Example Preparation

An injectable composition is prepared by mixing 10 mg each of: HA,TSG-6, PTX-3, and TSP-1, each of which is obtained from a commercialsource, with 100 mg of a preparation comprising: placental tissue,umbilical cord tissue, amniotic membrane tissue, chorion tissue orcombinations thereof; and then mixed with 10 mL of 0.9% sterile saline.The mixture is incorporated into a dosage unit form suitable foradministration by injection.

Example 2: Characterization of Amniotic Membrane Components

Material and Methods

The concentration of proteins in each extract was quantitated by the BCAProtein Assay Kit (Pierce, Rockford, Ill.). The concentration ofhyaluronic acid (HA) in each extracts was assayed with Hyaluronic Acid(HA) Quantitative Test Kit (Corgenix, Westminster, Colo.) based on ELISAusing a standard curve provided by the manufacturer prepared by serialdilution of HA.

HA Molecular Weight Range Analysis by Hyaluronidase Digestion

The HA molecular weight ranges of the extracts were analyzed by agarosegel electrophoresis according to the method described by Lee and Cowman(Lee H. G. and Cowman, M. K. An Agarose Gel Electrophoretic Method forAnalysis of Hyaluronan Molecular Weight Distribution. AnalyticalBiochemistry, 1994, 219, 278-287). The samples were subjected to 0.5%agarose gel electrophoresis followed by staining using 0.005% Stains-All(Sigma, cat #23096-0) in 50% ethanol. The gel was stained overnightunder a light-protective cover at room temperature (Shorter stainingperiods of 3-4 hr can also give acceptable results). HA was visualizedas blue bands after destaining by transferring the gel to H₂O andexposed to the room light for approximately 6 hr. The molecular weightstandards included λ DNA-BstE II digested restriction fragments (cat#D9793, Sigma) ranging in MW from 0.9 to 5.7×10⁶. The authenticity of HAwas further verified by incubation of the extract with or without 10units/ml hyaluronidase (Sigma #H1136) in the reaction buffer (50 mMTris-HCl, pH7.5, 0.1 M NaCl, 1% Triton X-100, 0.1% BSA supplemented withthe above protease and phosphatase inhibitors) for 2 h at 37° C. using apositive control of high MW HA (cat #H1876, Sigma) purified from humanumbilical cords.

Western Blot Analyses

The above extracts were electrophoresed on 4-15% denatured acrylamidegels and transferred to the nitrocellulose membrane, and thenimmunoblotted with a rabbit antihuman inter-α-trypsin inhibitor (rabbitpolyclonal antibody (cat #A0301, DAKO at 1:1000), a rabbit anti-humanTSG-6 polyclonal antibody (provided by Dr. Tony Day at 1:1000 dilution),a rat monoclonal anti-PTX3 antibody (Alexis Biochemicals, ALX-804-464, 1μg/ml), an anti-thrombospondin-1 antibody obtained from Calbiochem (Cat#BA24), and a goat anti-human Smad 7 antibody (AF2029, 1:1000, R & DSystems). Imunoreactive protein bands were detected by Western Lighting™Chemiluminesence Reagent (PerkinElmer).

Results

Experiments showed that the observed suppressive effect on the TGF β1promoter activity was abolished when water-soluble AM extracts werepre-heated at 90° C. for 10 minutes, suggesting that the responsiblecomponent(s) most likely contained protein(s), of which the conformationis important.

Quantitation of HA and Proteins in AM Extracts

The results, summarized Table 1, showed that all AM and jelly extractscontained both HA and proteins. In general, the weight ratio betweenproteins and HA was high in the Total Extract than the supernatant(e.g., L and H for PBS, and A for Buffer A) after centrifugation for AM,suggesting that most protein-containing materials were eliminated bycentrifugation. However, this trend was not noted in AM Jelly,suggesting that AM extracts contained more proteins than Jelly (see Tunder PBS and T under A/B/C). The ratio between proteins and HA was alsoincreased from Extract A to Extracts B and C for both AM and AM jelly,further supporting that HA was mostly present in the soluble form, andvice versa proteins were found more in the water-insoluble components.Furthermore, HA was largely removed from AM Jelly after centrifugationin A/B/C.

TABLE 1 Tissue AM Jelly Buffer PBS A/B/C PBS A/B/C Fraction T L H T A BC T L H T A B C Protein 8645 1370 1467 8645 2731 930 2698 3836 3645 35893836 3893 527 1364 (μg/ml) HA 75 62 44 60 74 7 35 80 90 96 129 94 2 7(μg/ml) Protein/HA 115 22 33 144 37 133 77 48 41 37 30 41 264 195[Note]: T: Total; L: the supernatant following the low speedcentrifugation of the total extract; H: the supernatant following thelow speed centrifugation of the total extract; A, B, C: Extracts, seetext.

HA in Different AM Extracts had Molecular Weights Greater than OneMillion Daltons

High molecular weight (>10⁶ daltons) of HA was present in the totalextracts and Extract A (FIG. 10). However, even higher MW of HA waspresent in Extract B, while HA was found in a narrow band with evenhigher MW in Extract C (FIG. 10). All of the HA-containing componentsdisappeared after hyaluronidase digestion, confirming that they indeedcontained HA. Compared to the positive control of HA obtained from Sigma(cat #H1136), a similar high molecular weight (>10⁶ daltons) of HA wasalso found in both supernatants obtained after low and high speeds ofcentrifugation (FIG. 11). Again these HA-containing bands disappearedafter hyaluronidase digestion. A similar result was obtained for AMjelly.

Inter-α-Trypsin Inhibitor (IαI) was Present in Different AM Extracts andits Heavy Chains (HCs) were Covalently Linked with HA

FIG. 12 showed that before digestion with hyaluronidase, free heavychains were present in different complexes, and a small amount of lightchain was also present (UTI or bikunin). However, in all extracts, i.e.,total and Extracts A, B, and C, there was also a covalently linkedcomplex between HA and heavy chains of IαI as the latter was releasedonly after hyaluronidase digestion. The same result was obtained inExtracts H and L obtained by two different speeds of centrifugation(FIG. 13).

Tumor Necrosis Factor-Stimulated Gene 6 (TSG-6) was Also Present in AMExtracts

FIG. 14 showed that TSG-6 (about 38 kDa) was present in Total, Extract Aand Extract C. In Extract A, there was a band of about 38 kDa migratedclose to that of the purified TSG-6 (35 kD). The identity of other bandsof about 45 and 55 kDa was unknown. Total AM extract (withoutcentrifugation) “T” showed two bands (both above 35 kD), and the higherone (55 kD) that were found in Extract A (after centrifugation), whilethe lower one (45 kD) was found in Extract C. All of these bands werenot significantly altered when samples were treated with hyaluronidase(FIG. 14) or with F-glycosidase (FIG. 15). However, digestion withchondroitin sulfate ABC lyase resulted in more noticeable 38 kD bandusing antibody RAH-1 (FIG. 16) but not using antibody MAB2104 (FIG. 17).

Pentraxin-3 (PTX-3) was Exclusively Present in Water-Soluble AM Extractsand Formed a Complex with HA

FIG. 18 showed that PTX3 was also present in AM extracts and wascomplexed with HA in the water soluble extract A only.

Thrombospondin-1 (TSP-1) was Present in Different AM Extracts

FIG. 19 showed that all AM extracts had a high molecular weight band ofTSP-1 while the total extract (T) and Extract C also had some bandsbetween 35-120 kDa. Hyaluronidase digestion did not change the reactivepattern except some bands became a little stronger or weaker.

Smad7 was Present in Mostly in Water-Insoluble AM Extracts

Smad 7 was found in both PBS extracts and urea extracts of AM (FIG. 20).

Example 3: Signaling Pathways Control Proliferation and EMT ofEpithelial Cells

Proliferation and EMT by dysfunctional epithelial cells are two majorpathological processes. During rhegmatogenous retinal detachments(RRDs), retinal pigment epithelium (RPE) cells are dispersed into thevitreous, which contains many growth factors and cytokines (e.g., EGF,FGF, PDGF, TGF-β, VEGF and IFN-γ) necessary for the bioactivity ofproliferative vitreoretinopathy (PVR) as identified recently. Tounderstand how growth factors might contribute to proliferation and EMTof dispersed RPE cells, an in vitro culturing model of ARPE-19 cells,which these cells exhibit contact inhibition after seven days ofpost-confluence was used.

Following perturbation of contact inhibition by EGTA, cell proliferation(BrdU labeling) and EMT (loss of normal RPE phenotype markers ofN-cadherin, ZO-1, Na, K-ATPase, and RPE65, and express of mesenchymalphenotype markers of vimentin, S100A4, and α-SMA) were only induced inthe presence of EGF and/or FGF-2. This pathological process required theactivation of canonical Wnt signaling, as evidenced by the increasednuclear level and interaction of β-catenin and LEF, as well asupregulation of TCF/LEF transcriptional activation. The activation ofcanonical Wnt signaling was confirmed by using a Wnt inhibitor XAV939and overexpression of constitutive active β-catenin (S33Y) in blockingor rescuing experiments. Addition of TGF-β1 also lead to EMT byactivating Smad/ZEB1/2 signaling, which suppressed proliferation andactivation of canonical Wnt signaling. Furthermore, canonical Wntsignaling triggered by EGF+FGF-2 was sufficient and synergized withTGF-β1 to lead to EMT (FIG. 1). These findings provided the mechanisticinsight for us to target these two signaling pathways so as to preventPVR. The in vitro model using ARPE-19 cell line was further optimizedbased on a low cell density instead of by adding EGTA to confluent cellsto initiate proliferation to better mimic PVR. The results showedHC-HA/PTX3 did not harm the non-stimulated ARPE-19 cells (FIG. 5A), butsignificantly suppressed the proliferation (FIGS. 5B and 5C) and thenuclear localization of phosphorylated Smad2/3 (FIGS. 6A and 6B) afterstimulation with EGF+FGF-2 and EGF+FGF-2+TGF-β1, respectively. Theestablishment of such an in vitro model allowed for the determination ofoptimal dosing of HC-HA/PTX3 to be used for in vivo testing, in therabbit PVR animal model provided herein (FIGS. 7A-D).

Example 4: Development of an Animal Model of PVR

PVR was successfully reproduced in rabbits (see FIGS. 7A-7D) by vitreousdetachment by gas compression vitrectomy followed by intravitrealinjection of rabbit RPE cells to mimic human PVR. Rabbits were chosenbecause they can develop medullary wing detachments that simulateretinal detachments in humans and show PVR-like features.

NZW rabbits (Female, aged 3-7 months, weighing between 1.5 and 5.0 kg)were subjected to vitrectomy by intravitreal gas injection by injecting0.3 ml of 100% C3F8 gas into the vitreous cavity using a 32 gauge ½″needle 3 mm posterior to the corneoscleral limbus under directvisualization using indirect ophthalmoscopy following anterior chamberparacentesis performed to lower the intraocular pressure and reduce thepossibility of ocular damage caused by an acute increase in pressure.Indirect ophthalmoscopy was performed to ensure there is normal vascularflow in the retina. The intraocular pressure was checked using a Perkinstonometer until the intraocular pressure is below 20 mmHg. PVR wascreated by intravitreal injection of 2.0×10⁵ rabbit RPE cells that hadbeen prepared from tissue cultured homologous primary rabbit RPE cellsin a total of 0.1 ml volume via a 32 gauge ½″ needle, with the bevelfacing upward, and injected into the vitreous cavity, just in front ofthe optic nerve head (slowly, to prevent retinal damage). If thetreatment of HC-HA/PTX3 was simultaneous with RPE cells, then PBS or twodifferent doses of HC-HA/PTX3 were injected similarly into the vitreouscavity of the control rabbits or treated rabbits, respectively. If thetreatment of HC-HA/PTX3 was subsequent with RPE cells, PBS andHC-HA/PTX3 was injected into the vitreous cavity one week later. In eachcondition, the rabbits were immediately placed on their backs for 1 h toallow the cells and reagents to settle over the vascular wings of theretina.

Four weeks after injection of the intravitreal HC-HA/PTX3 or saline therabbits were sacrificed by euthanasia by anesthetic overdose withEuthasol (390 mg/mL/kg, intravenous). The eyeball was enucleated withall conjunctival tissues, and fixed in 10% formalin. The eyes underwentexternal examination and then the superior cap is removed to allowinternal examination. The gross anatomical examination of the enucleatedeyes was photographed using a Nikon D600 camera (FIG. 7C and FIG. 7D).

Example 5: HC-HA/PTX3 is a Unique Matrix Component Responsible for AM'sTherapeutic Actions

HC-HA/PTX3 complex, first found in the cumulus-oocyte complexsurrounding the ovulated oocyte, plays a critical role in fertilization.HC-HA/PTX3 is abundantly present in human AM and this discovery has ledto several exciting findings: (1) AM epithelial and stromal cellsexpress all components (HA, HC1, HC2, bikunin, TSG-6, and PTX3)necessary for HC-HA/PTX3 biosynthesis (FIG. 2A); (2) HC-HA/PTX3 purifiedfrom AM extract (AME) consists of HMW HA (>3000 kDa) with covalentlylinked HC1 of IαI and tightly bound PTX3 (FIG. 2B-2D), but not HC2,bikunin, or TSG-6 and (3) HC-HA/PTX3 is responsible for AM's therapeuticactions which is briefly summarized below.

To make sure HC-HA/PTX3 prepared from each lot of AM donors wasconsistent biochemically and functionally, a manufacturing process usingoptimized SOPs under GMP facility was established. Although the yield ofHC-HA/PTX3 from different AM donors varied, no significant differencesin the potency assays were observed, which were developed based oninhibition of tartrate resistant acid phosphatase activity inosteoclasts and on promotion of macrophage M2 polarization inIFN-γ/LPS-stimulated macrophages. Consequently, the reference materialto validate the release of each lot of HC-HA/PTX3 to be used in in vitroand in vivo studies was established.

Inflammation involving neutrophils and macrophages plays an importantrole in PVR development. Injection of macrophages into the rabbitvitreous induced epiretinal membranes, posterior vitreous separation,and retinal detachment. Macrophages can transdifferentiate intofibroblast-like cells and secrete growth factors (e.g., PDGF), whichcontribute to proliferation and EMT of RPE cells, the two key events inPVR pathogenesis. Soluble HC-HA/PTX3, but not HA, significantly promotedapoptosis of activated (by fMLP or LPS) but not resting neutrophils.Similarly, soluble HC-HA/PTX3, but not HA, dose-dependently promotedapoptosis of activated (by IFN-γ, LPS or IFN-γ/LPS) but not restingmacrophages. In addition, soluble and immobilized HC-HA/PTX3, but notHA, promoted phagocytosis of apoptotic neutrophils by macrophages.Immobilized HC-HA/PTX3 promoted anti-inflammatory M2 polarization ofLPS- or IFN-γ/LPS-activated macrophages. In addition, such M2polarization was coupled with notable downregulation of IL-23, which wasproduced by activated macrophages and dendritic cells to activate Th17cells. Consequently, subconjunctival injection of HC-HA/PTX3 prolongedsurvival of allogeneic corneal transplants in mice. These data supportthe notion that HC-HA/PTX3 is a novel complex which can suppressinflammation mediated by both neutrophils and macrophages.

Example 6: HC-HA/PTX3 Downregulated Canonical Wnt Signaling in HumanLimbal Epithelial Progenitor and Niche Cells

AM inhibited squamous metaplasia of human conjunctival epithelium bydownregulating the expression, phosphorylation, and nucleartranslocation of β-catenin. Furthermore, HC-HA/PTX3 downregulatedcanonical Wnt signaling in human limbal epithelial progenitor cells(LEPCs) and niche cells (LNCs). Specifically, immobilized HC-HA/PTX3upregulated transcript expression of non-canonical but not canonical Wntligands (e.g., Wnt 2B, Wnt 3A, Wnt 5A, Wnt 5B, Wnt7A), Wnt negativeregulators, and planer cell polarity (PCP) factors in LEPCs/LNCs asmeasured by Wnt Signaling Pathway RT² Profiler PCR Array Plate (FIG.3A). The immunostaining data further confirmed that immobilizedHC-HA/PTX3 prevented the nuclear translocation of β-catenin as shown inthe positive control cells seeded in 3D Matrigel. In contrast,transcript expression (FIG. 3A) and nuclear translocation (FIG. 3B) ofC-JUN, a key player of non-canonical Wnt (PCP) signaling, was noted inLNCs when seeded on immobilized HC-HA/PTX3 but not 3D Matrigel (FIG.3A). Note that activation of non-canonical Wnt (PCP) signaling is knownto suppress that of canonical Wnt signaling.

Example 7: HC-HA/PTX3 Downregulates Canonical TGF-β1/Smad Signaling inHuman Corneal Fibroblasts (HCF)

It has been reported that expression of TGF-β1,2,3 and TGF-βRIItranscripts (using Northern blot) is downregulated in HCF and humanlimbal and conjunctival fibroblasts cultured on the AM stroma. AMEinduced cell aggregation and prevents expression of α-SMA bymyofibroblasts. Human and mouse keratocytes seeded on AM stromamaintained their normal phenotype without eliciting nucleartranslocation of pSmad2/3 even if they were exposed to serum or TGF-β1.Soluble HC-HA/PTX3 suppressed the TGF-β1 promoter activity of HCF (FIG.4A). It is known that exogenous TGF-β1 expectedly upregulates TGF-β1,but not TGF-β2 (FIG. 4B), in HCF seeded on both plastic and immobilizedHA. However, TGF-β1 upregulation was not observed on immobilizedHC-HA/PTX3. Surprisingly, TGF-β3, an anti-scarring isoform, wasupregulated only by HC-HA/PTX3, with or without TGF-β1 (FIG. 4C).Expression of TGF-βRII was reduced to nearly nil on HC-HA/PTX3 afterTGF-β1 challenge (FIG. 4D). As expected, exogenous TGF-β1 caused thenuclear translocation of pSmad2/3 (FIG. 4E) and positive cytoplasmicexpression of α-SMA (FIG. 4F) in HCF on plastic and HA. However,HC-HA/PTX3 effectively blocked these TGF-β1-induced changes in HCF.Collectively, HC-HA/PTX3 downregulated canonical TGF-β1 signaling andprevented myofibroblast differentiation triggered by exogenous TGF-β1 inHCF.

Example 8: Preparation of Preserved Human Fetal Support Tissue

Human placenta was collected at elective cesarean delivery. The placentawas flattened onto nitrocellulose paper (Hybond N+, Amersham, England),with the epithelium surface up. The fetal support tissue samples werestored at −80° C. in DMEM/glycerol 1:2 (v/v) until use.

Example 9: Amniotic Membrane Extract Preparations

Fresh and frozen human placentas were obtained from Bio-tissue, Inc.(Miami, Fla.). The entire procedure for preparation of total human AMextracts (AME) was carried out aseptically so as to be used forsubsequent cell culture-based experiments. The AM was sliced into smallpieces to fit into the barrel of a BioPulverizer (Biospec Products,Inc., Bartlesville, Okla.), frozen in the liquid nitrogen, pulverizedinto a fine powder, and weighed. Cold 1×PBS buffer, pH 7.4, containingprotease inhibitors (protease inhibitor cocktail, P8340, Sigma, andsupplemented with 1 mM PMSF) and phosphatase inhibitors (50 mM sodiumfluoride and 0.2 mM sodium vanadate) was added to the powder at 1:1(ml/g). The mixture was kept on ice and homogenized with a Tissue Tearor(Biospec Products, Inc., Dremel, Wis.) 5 times, 1 minute each, with a 2minute cooling interval. These water-soluble extracts were designated as“Total” AM extracts (AME).

Total AM extracts were divided into two 50 ml conical centrifuge tubes.One was centrifuged at high speed (HS, 48,000×g) and the other one wascentrifuged at a low speed (LS, 15,000×g) at 4 C° Aliquots of the HS andLS supernatant were transferred to sterile 1.5 ml Eppendorf tubes andwere designated as AM/HS and AM/LS, respectively. Desired AM/HS sampleswere frozen at −20 C° for 1 h before lyophilization. The samples werethen placed in the chamber of FreeZone (Labconco, Kansas City, Mo.) withholes on the cap. Samples were lyophilized at −50 C° at a vacuum of 0.85mBar for 5 hours. Before use, the samples were reconstituted with thesterile distilled H₂O to the same volume. The same method was also usedto prepare extracts from AM jelly, which was easily scraped from theadherent material on the AM stroma.

Example 10: Total Soluble Human Amniotic Membrane and Amniotic MembraneJelly Extract Preparations

Frozen human placenta material was obtained from Bio-Tissue, Bio-tissue,Inc. (Miami, Fla.). The entire procedure for preparation of total humanAM extracts (AME) was carried out aseptically so as to be used forsubsequent cell culture-based experiments. The AM was sliced into smallpieces to fit into the barrel of a BioPulverizer (Biospec Products,Inc., Bartlesville, Okla.), frozen in the liquid nitrogen, pulverizedinto a fine powder, and weighed. Cold 1×PBS buffer, pH 7.4, containingprotease inhibitors (protease inhibitor cocktail, P8340, Sigma, andsupplemented with 1 mM PMSF) and phosphatase inhibitors (50 mM sodiumfluoride and 0.2 mM sodium vanadate) was added to the powder at 1:1(ml/g). The mixture was kept on ice and homogenized with a Tissue Tearor(Biospec Products, Inc., Dremel, Wis.) 5 times, 1 minute each, with a 2minute cooling interval. These water-soluble extracts were designated as“Total” AM extracts (AME).

The total water-soluble extract was mixed for 1 hr at 4 C°, subsequentlyfractionated by two different speeds of centrifugation at 4° C. for 30min, i.e., 15000×g and 48000×g, and the resultant supernatant wasdesignated as L and H, respectively. Each supernatant was divided intoaliquots and stored at −80° C. This method was also used to prepareextracts from AM jelly, which was easily scraped from the adherentmaterial on the AM stroma.

Example 11: Total Soluble Human Amniotic Membrane and Amniotic MembraneJelly Extracts by Different Buffers and Fractionation Methods

In examining preparations in different extraction buffers, the powder asprepared from above was weighed and mixed with Buffer A (Isotonic Lowsalt): 100 mM Tris-HCl, pH 7.6, 150 mM NaCl, 4 mM EDTA, 1% Triton X-100at the wet weight (g) of AM to the buffer (ml) at 1:1 ratio by stirringat 4° C. for 1 hr. After centrifugation at 48000×g, the resultant pelletwas subsequently extracted by Buffer B (high salt): 100 mM Tris-HCl, pH7.6, 1 M NaCl, 4 mM EDTA, 1% Triton X-100 by stirring at 4 C for 1 hr.Again after centrifugation at 48000×g, the pellet was finally extractedby Buffer C (4 M guanidine hydrochloride): 100 mM sodium acetate, pH5.8, 4 M guanidine hydrochloride, 4 mM EDTA, 1% Triton X-100 by stirringat 4° C. for 24 hr. All the above three buffers were supplemented withthe following protease and phosphatase inhibitors: 1 μg/ml aprotinin, 1μg/ml leupeptins, 1 μg/ml pepstatin A, 0.5 mM PMSF, 50 μM sodiumfluoride and 0.2 μM sodium vanadate. The resultant supernatants,designated as Extract A, B, and C, respectively, were dialyzed againstthe dialysis buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl) supplementedwith 0.5 mM PMSF at 4° C. for 6 hr and dialysis buffer was changedtwice, each with 500×(the volume ratio of dialysis buffer:samples).After dialysis, the volume of each sample was measured and adjusted tothe same volume with the dialysis buffer. The same method was also usedto prepare extracts from AM jelly, which was the adherent material onthe AM stroma that could be easily scraped off.

Example 12: Preparation of Total Soluble Human Amniotic MembraneExtracts in PBS

The entire procedure for preparation of total soluble human AM extracts(T) was carried out aseptically so as to be used for subsequent cellculture-based experiments. Frozen human placenta was obtained fromBio-Tissue, Inc. (Miami, Fla.), from which AM was retrieved. AM wassliced into small pieces to fit into the barrel of a BioPulverizer(Biospec Products, Inc., Bartlesville, Okla.), frozen in the liquidnitrogen, and then pulverized into a fine powder. The powder was weighedand mixed with cold PBS buffer (prepared by adding distilled H₂O to1×PBS, pH7.4, from 10×PBS, cat #70011-044, Invitrogen, Carlsbad, Calif.)with protease inhibitors (protease inhibitor cocktail, P8340, Sigma, andsupplemented with 1 mM PMSF) and phosphatase inhibitors (50 mM sodiumfluoride and 0.2 mM sodium vanadate) at 1:1 (ml/g). The mixture was kepton ice and homogenized with a Tissue Tearor (Biospec Products, Inc.,Dremel, Wis.) for 5 times, 1 min each with a 2 min interval cooling.This water-soluble extract was designated as “Total” (T). The totalwater-soluble extract was mixed for 1 hr at 4° C., centrifuged at 4° C.for 30 min at 48000×g. The supernatant was divided into aliquots andstored at −80° C.

Example 13: Preparation of Water-Soluble AM Stromal Extracts

Using aseptic techniques, frozen human AM obtained from Bio-Tissue, Inc.(Miami, Fla.) was briefly washed 2-3 times with HBSS to remove theoriginal storage medium. The AM stroma was scraped by spatula, frozen inthe air phase of liquid nitrogen and grounded to fine particles byBioPulverizer (Biospec Products, Inc., Bartlesville, Okla.) followed byhomogenization on ice with Tissue Tearor (Biospec Products, Inc.,Dremel, Wis.) in PBS, pH 7.4, for 1 min. The homogenate was mixed byrotation for 1 h and centrifuged at 14,000×g for 30 min at 4° C. Thesupernatant in PBS was then collected, and stored in aliquots at −80° C.The protein concentration was determined by BCA assay. Thiswater-soluble protein extract, designated as amniotic stromal extract(ASE), was used for experiments described herein.

Example 14: AM Stromal Extract Preparation

The complete procedure for preparation of protein extracts was carriedout aseptically. Frozen human AM obtained from Bio-Tissue (Miami, Fla.)was briefly washed 2-3 times with HBSS (Invitrogen, Carlsbad, Calif.) toremove the storage medium. AM stroma was scraped from the stromal sideof the AM by spatula for AM stroma extract preparation. Human placentaas well as chorion obtained from Baptist Hospital (Miami, Fla.) wasrinsed 3 times with HBSS to remove blood. To prepare the water-solubleprotein extract, total AM, scraped AM stroma, stroma-removed AM,placenta, and chorion were each frozen in the air phase of liquidnitrogen and each ground to fine particles using a BioPulverizer(Biospec Products, Inc., Bartlesville, Okla.) followed by homogenizationon ice with Tissue Tearor (Biospec Products, Inc., Dremel, Wis.) in PBS(pH 7.4) for 1 min. Each homogenate was mixed for 1 h and centrifuged at14,000 g for 30 min at 4° C. Each supernatant (in PBS) was thencollected and stored in aliquots (0.5 ml) at −80° C. The BCA assay(Pierce, Rockford, Ill.) was used to quantitate the total protein indifferent extracts.

Example 15: Preparing Water-Soluble and Lyophilized Powder Forms ofHuman AM Extracts

To prepare human AM extracts, the entire procedure was carried outaseptically. Unless otherwise noted, the AM extracts were handled atroom temperature during the steps of the procedure. First, fresh orfrozen human AM was obtained, preferably from Bio-Tissue, Inc. (Miami,Fla.). Frozen AM was briefly washed 2-3 times with HBSS (Invitrogen,Carlsbad, Calif.) to remove the storage medium. Fresh human placenta orchorion was rinsed 3 times with HBSS to remove blood.

To prepare the water-soluble form of AM extracts, the AM (e.g., AMstroma, stroma-removed AM, placenta, chorion) was transferred to asterile 50 ml centrifuge tube and centrifuged at 4° C. for 5 min at5000×g to remove the excess fluid. The AM was weighed, transferred to a100 mm or 150 mm sterile Petri dish, and frozen in the air phase of aliquid nitrogen container for 20 min to facilitate the subsequenthomogenization. The frozen AM was then sliced into small pieces with adisposable scalpel or ground to fine particles using a BioPulverizer(Biospec Products, Inc., Bartlesville, Okla.) or other suitable device,and homogenized with Tissue Tearor (Biospec Products, Inc., Dremel,Wis.), or other suitable device, in phosphate buffered saline (PBS) orDMEM without phenol red (Invitrogen, Carlsbad, Calif.) at neutral pH.For biochemical characterization and purification, the above solutionswere supplemented with the following proteinase inhibitors: 1 μg/mlaprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 mM PMSF.However, if the extract was to be directly added to cell culture, noprotease inhibitors were added. The homogenate was mixed at 4° C. for 30min and centrifuged at 15,000×.g for 30 min. The supernatant (i.e., AMextract) was collected and stored in aliquots (0.5 ml) at −80° C. TheBCA assay (Pierce, Rockford, Ill.) was used to quantitate the totalprotein in each AM extract.

To prepare the lyophilized powder form of AM extracts, frozen AM wasground to fine particles using a BioPulverizer (Biospec Products, Inc.,Bartlesville, Okla.), or other suitable device, and further homogenizedas described herein. Aliquots of approximately 0.5 ml were lyophilizedby SpeedVac (Savant Instruments Inc., Farmingdale, N.Y.) at 4° C. for 4h to decrease the weight from 280 mg to 32 mg (about 89% reduction). Thelyophilized powder was weighed and stored at −80° C. Before use, thelyophilized powder was reconstituted in a suitable buffer.

To prepare AM stromal extracts, the AM stroma was scraped from thestromal surface of intact total AM leaving the basement membrane and theamniotic epithelium intact, and the frozen AM stroma was ground using aBioPulverizer as described herein. The stroma was extracted with PBS ata neutral pH at 4° C. for 30 min and centrifuged at 15,000×g for 30 min.The supernatant was collected and stored in aliquots (0.5 ml) at −80° C.The BCA assay (Pierce, Rockford, Ill.) was used to quantitate the totalprotein in the AM stromal extract.

Example 16: Suppression of TGF-β1 Promoter Activity

The fetal support preparations and compositions described hereinsuppress of TGF-β1 promoter activity as shown herein; thus the fetalsupport preparations and compositions described herein can be used foranti-scarring, anti-inflammatory, and anti-angiogenic therapies. Thefetal portion of the frozen amniotic membrane has a significantly higheranti-scarring effect than that of fresh amniotic membrane; the placentalportion of the frozen amniotic membrane also has a significantly higheranti-scarring effect than the fresh amniotic membrane. Therefore, thefrozen fetal support tissue, either the placental or fetal portion,showed more potent suppressive effects in TGF-β than the fresh fetalsupport tissue. This suppressive effect mediated by total fetal supporttissue extract obtained from frozen fetal support tissue wasdose-dependent over a range of 0.4 to 125 μg/ml (FIG. 8). Furthermore,such a suppressive effect could not be substituted by high MW HA alone(exceeding 100× of equivalent AM extract), and was lost after digestionwith hyaluronidase (FIG. 9), suggesting that it was mediated by acomplex between HA-IαI. Centrifugation at low or high speed did notaffect the suppressive effect significantly. However, subsequentlyophilization and reconstitution produced a more potent suppressiveeffect. Additionally, the overall suppressive effect of AM was morepotent than that of AM jelly.

Example 17: Fetal Support Tissue Preparations and Purified CompositionsUsed to Culture Cells

To examine the effect of fetal support tissue on the celldifferentiation process, myofibroblasts differentiated from AMSCs atpassage 2 were subcultured onto the stromal matrix of AM, and comparedto those subcultured on collagen I-coated dish as a control. After 7days of cultivation in DMEM with 10% FBS, AMSCs on collagen I stillmaintained a myofibroblastic shape. In contrast, cells seeded on fetalsupport tissue stromal matrix exhibited a mixture of round, spindle,elongated, and dendritic shapes. Thus, in some embodiments, fetalsupport tissue preparations have dedifferentiation abilities, and areused to slow cell differentiation.

Example 18: Effect of HC-HA/PTX3 on Cell Migration and Collagen GelContraction

Cell Culture and Treatment

ARPE-19, a human diploid RPE cell line, was cultured in HEPES-bufferedDMEM and Ham's F-12 (1:1) supplemented with 10% FBS, 50 units/mlpenicillin, and 50 μg/ml streptomycin at 37° C. in humidified air with5% CO₂. For post-confluence experiments, cells were continuouslycultured for 7 days upon 100% confluence before being tested. For lowcell density assays, cells were seeded at 1×10⁴/cm² or other densitiesovernight (20-24 h) followed by treatment with growth factors andcytokines for 24-120 h or 48 h (after optimization). In the case ofserum starvation, cells were incubated in serum-free (SF) medium for 24h followed by treatment with growth factors and cytokines for 24-120 h.BrdU (10 μM) labeling was performed for 4 h prior to the termination ofthe growth factors/cytokines treatment.

Purification of HC-HA/PTX3 from Human AM

HC-HA/PTX3 was prepared from cryopreserved human placentas provided byBio-Tissue, Inc. (Miami, Fla.). AM from the same donor was extracted byPBS (pH 7.4) to generate the PBS extract as reported. The extract wasthen fractionated by ultracentrifugation in a CsCl gradient at aninitial density of 1.35 g/ml in 4 M GnHCl at 35,000 rpm for 48 h at 15°C. (LM8 model, SW41 rotor, Beckman Coulter, Indianapolis, Ind.). A totalof 12 fractions (1 ml/fraction) was collected from each ultracentrifugetube. The weight of each fraction was measured to calculate the density.After the biochemical analysis (HA ELISA, BCA protein assay, and Westernblot, see below), fractions containing HA but little or no proteins werepooled and subjected to the second run of ultracentrifugation in a CsClgradient at an initial density of 1.40 g/ml. Selective fractions(containing HA but undetectable proteins measured by BCA assay anddesignated as HC-HA/PTX3) were pooled and dialyzed against distilledwater, lyophilized, and stored at −80° C. Therefore, the amount ofHC-HA/PTX3 was expressed based on the HA amount present in the complex.

Cell Migration

The migration assay was performed in 24-well transwell plate (8 μm poresize, Costar, Kennebunk, Me.) when 0.5 ml DMEM/F12 (1:1) without or withEGF (10 ng/ml), FGF-2 (20 ng/ml), and TGF-β1 (10 ng/ml) was added in thelower compartment while 0.1 ml of ARPE-19 cell re-suspended in DMEM/F12(2×10⁶/ml) treated with PBS (vehicle control), HA (25 μg/ml), orHC-HA/PTX3 (25 μg/ml) was added to the upper compartment. Afterincubation at 37° C. for 4 h, cells not migrating through the pores wereremoved by a cotton swap, while cells on the filter facing the lowercompartment were fixed with 5% glutaraldehyde, stained with 1% crystalviolet, and counted from six random microscopic fields for each controlor treatment group.

Collagen Gel Contraction

0.25 ml of collagen type I solution (Corning, Bedford, Mass.) in coldDMEM/F12 (2.5 mg/ml) was added to each well of 24-well plates, followedby incubation at 37° C. for 1 h before adding 0.5 ml of ARPE-19 cells orprimary human RPE cells (each at 5×10⁵/ml) without or with TGF-β1 (10ng/ml) and treatment of PBS (vehicle control), HA (25 μg/ml), orHC-HA/PTX3 (25 μg/ml) on the top of collagen gel. After 24 h, the gelswere freed from the walls of the culture wells with a small spatula. Thephotographic images of collagen gels were digitalized and the area wasmeasured with NIH ImageJ 1.45 software. The percentage of gelcontraction was determined by measuring the gel size at 72 h whencompared to the initial size (at 0 h) and compared among groups.

Results

HC-HA/PTX3 (25 μg/ml) as well as HA (25 μg/ml) completely suppressedmigration of ARPE-19 cells under the stimulation of EGF (10 ng/ml),FGF-2 (20 ng/ml), and TGF-β1 (10 ng/ml) (FIG. 21). In contrast,HC-HA/PTX3, but not HA, significantly reduced the TGF-β1-inducedcollagen gel contraction in both ARPE-19 cells and primary human RPEcells (FIG. 22).

While preferred embodiments have been shown and described herein, itwill be obvious to those skilled in the art that such embodiments areprovided by way of example only. Numerous variations, changes, andsubstitutions may now occur. It should be understood that variousalternatives to the embodiments described herein can be employed inpracticing the described methods. It is intended that the followingclaims define the scope of the embodiments and that methods andstructures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. An composition for preventing or reducingproliferation, cell migration, or epithelial-mesenchymal transition(EMT) of epithelial cells, comprising: (a) a preparation of fetalsupport tissue; and (b) a pharmaceutically acceptable diluent,excipient, vehicle, or carrier.
 2. The composition according to claim 1,wherein the fetal support tissue is selected from placenta, placentalamniotic membrane, umbilical cord, umbilical cord amniotic membrane,chorion, amnion-chorion, amniotic stroma, amniotic jelly, or acombination thereof.
 3. The composition of claim 1, wherein the fetalsupport tissue is frozen or previously frozen.
 4. The compositionaccording to claim 1, wherein the epithelial cells are selected fromretinal pigment epithelial cells (RPE), conjunctival epithelial cells,corneal epithelial cells, limbal epithelial cells, and renal epithelialcells.
 5. The composition according to claim 1, wherein the epithelialcells are human epithelial cells.
 6. The composition of claim 1, whereinthe preparation of fetal support tissue is an extract of fetal supporttissue, a homogenate, a powder, morselized fetal support tissue,pulverized fetal support tissue, ground fetal support tissue, purifiedHC-HA/PTX3, or a combination thereof.
 7. The composition according toclaim 1, wherein the composition is a gel, a solution, or a suspension.8. The composition according to claim 1, wherein the preparationcomprises HC-HA/PTX3.
 9. The composition according to claim 1, whereinthe composition is for local administration.
 10. The compositionaccording to claim 1, wherein the composition is formulated forintraocular injection, subretinal injection, intravitreal injection,periocular injection, subconjunctival injection, retrobulbar injection,intracameral injection, or sub-Tenon's injection.
 11. An injectablecomposition for treating or preventing Proliferative Vitreoretinopathy(PVR) consisting essentially of: (a) substantially isolated HC-HA/PTX3,reconstituted HC-HA/PTX3, or a combination thereof; and (b) apharmaceutically acceptable diluent, excipient, vehicle, or carrier;wherein the composition is suitable for injection.
 12. The compositionaccording to claim 11, wherein the HC-HA/PTX3 is isolated from a fetalsupport tissue, wherein the fetal support tissue is placenta, placentalamniotic membrane, umbilical cord, umbilical cord amniotic membrane,chorion, amnion-chorion, amniotic stroma, amniotic jelly, or acombination thereof.
 13. The composition of claim 12, wherein the fetalsupport tissue is frozen or previously frozen.
 14. The composition ofclaim 11, wherein the composition is in an amount effective forpreventing or reducing the proliferation, cell migration or EMT ofepithelial cells.
 15. The composition of claim 14, wherein theepithelial cells are retinal pigment epithelial cells (RPE).
 16. Thecomposition of claim 14, wherein the epithelial cells are humanepithelial cells.
 17. The composition of claim 11, wherein thesubstantially isolated HC-HA/PTX3 is isolated from fetal support tissueby ultracentrifugation.
 18. The composition according to claim 11,wherein the composition is a gel, a solution, or a suspension.
 19. Thecomposition according to claim 11, wherein the composition is formulatedfor intraocular injection, subretinal injection, intravitreal injection,periocular injection, subconjunctival injection, retrobulbar injection,intracameral injection or sub-Tenon's injection.
 20. An injectablecomposition for treating or preventing Proliferative Vitreoretinopathy(PVR) consisting essentially of: (a) substantially isolated HC-HA/PTX3,reconstituted HC-HA/PTX3, or a combination thereof; (b) an additionaltherapeutic agent; and (c) a pharmaceutically acceptable diluent,excipient, vehicle, or carrier; wherein the composition is suitable forinjection.
 21. An injectable composition for treating or preventingProliferative Vitreoretinopathy (PVR) comprising: (a) a preparation offetal support tissue comprising HC-HA/PTX3 and at least one othercomponent of fetal support tissue; and (b) a pharmaceutically acceptablediluent, excipient, vehicle, or carrier; wherein the composition issuitable for injection.
 22. The composition according to claim 21,wherein the fetal support tissue is placenta, placental amnioticmembrane, umbilical cord, umbilical cord amniotic membrane, chorion,amnion-chorion, amniotic stroma, amniotic jelly, amniotic fluid, or acombination thereof.
 23. The composition of claim 21, wherein the fetalsupport tissue is frozen or previously frozen.
 24. The composition ofclaim 21, wherein the composition is in an amount effective forpreventing or reducing the proliferation, cell migration or EMT ofepithelial cells.
 25. The composition according to claim 24, wherein theepithelial cells are retinal pigment epithelial (RPE) cells.
 26. Thecomposition of claim 21, wherein the preparation of fetal support tissueis an extract of fetal support tissue, micronized fetal support tissue,a homogenate, a powder, morselized fetal support tissue, pulverizedfetal support tissue, ground fetal support tissue, purified HC-HA/PTX3,or a combination thereof.
 27. The composition according to claim 21,wherein the composition is a gel, a solution, or a suspension.
 28. Thecomposition according to claim 21, wherein the fetal support tissue ishuman, non-human primate, bovine, or porcine.
 29. The compositionaccording to claim 21, wherein the composition is formulated forintraocular injection, subretinal injection, intravitreal injection,periocular injection, subconjunctival injection, retrobulbar injection,intracameral injection or sub-Tenon's injection.